Whole cell engineering by mutagenizing a substantial portion of a starting genome combining mutations and optionally repeating
First Claim
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1. A method for identifying proteins by differential labeling of peptides, the method comprising the following steps:
- (a) providing a sample comprising a polypeptide;
(b) providing a plurality of labeling reagents which differ in molecular mass that can generate differential labeled peptides that do not differ in chromatographic retention properties and do not differ in ionization and detection properties in mass spectrographic analysis, wherein the differences in molecular mass are distinguishable by mass spectrographic analysis;
(c) fragmenting the polypeptide into peptide fragments by enzymatic digestion or by non-enzymatic fragmentation;
(d) contacting the labeling reagents of step (b) with the peptide fragments of step (c), thereby labeling the peptides with the differential labeling reagents;
(e) separating the peptides by chromatography to generate an eluate;
(f) feeding the eluate of step (e) into a mass spectrometer and quantifying the amount of each peptide and generating the sequence of each peptide by use of the mass spectrometer;
(g) inputting the sequence to a computer program product which compares the inputted sequence to a database of polypeptide sequences to identify the polypeptide from which the sequenced peptide originated.
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Abstract
This invention relates to the field of cellular and whole organism engineering. Specifically, this invention relates to a cellular transformation, directed evolution, and screening method for creating novel transgenic organisms having desirable properties. Thus in one aspect, this invention relates to a method of generating a transgenic organism, such as a microbe or a plant, having a plurality of traits that are diffenentially activatable.
173 Citations
179 Claims
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1. A method for identifying proteins by differential labeling of peptides, the method comprising the following steps:
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(a) providing a sample comprising a polypeptide;
(b) providing a plurality of labeling reagents which differ in molecular mass that can generate differential labeled peptides that do not differ in chromatographic retention properties and do not differ in ionization and detection properties in mass spectrographic analysis, wherein the differences in molecular mass are distinguishable by mass spectrographic analysis;
(c) fragmenting the polypeptide into peptide fragments by enzymatic digestion or by non-enzymatic fragmentation;
(d) contacting the labeling reagents of step (b) with the peptide fragments of step (c), thereby labeling the peptides with the differential labeling reagents;
(e) separating the peptides by chromatography to generate an eluate;
(f) feeding the eluate of step (e) into a mass spectrometer and quantifying the amount of each peptide and generating the sequence of each peptide by use of the mass spectrometer;
(g) inputting the sequence to a computer program product which compares the inputted sequence to a database of polypeptide sequences to identify the polypeptide from which the sequenced peptide originated. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49)
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50. A method for defining the expressed proteins associated with a given cellular state, the method comprising the following steps:
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(a) providing a sample comprising a cell in the desired cellular state;
(b) providing a plurality of labeling reagents which differ in molecular mass but do not differ in chromatographic retention properties and do not differ in ionization and detection properties in mass spectrographic analysis, wherein the differences in molecular mass are distinguishable by mass spectrographic analysis;
(c) fragmenting polypeptides derived from the cell into peptide fragments by enzymatic digestion or by non-enzymatic fragmentation;
(d) contacting the labeling reagents of step (b) with the peptide fragments of step (c), thereby labeling the peptides with the differential labeling reagents;
(e) separating the peptides by chromatography to generate an eluate;
(f) feeding the eluate of step (e) into a mass spectrometer and quantifying the amount of each peptide and generating the sequence of each peptide by use of the mass spectrometer;
(g) inputting the sequence to a computer program product which compares the inputted sequence to a database of polypeptide sequences to identify the polypeptide from which the sequenced peptide originated, thereby defining the expressed proteins associated with the cellular state.
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51. A method for quantifying changes in protein expression between at least two cellular states, the method comprising the following steps:
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state;
(b) providing a plurality of labeling reagents which differ in molecular mass but do not differ in chromatographic retention properties and do not differ in ionization and detection properties in mass spectrographic analysis, wherein the differences in molecular mass are distinguishable by mass spectrographic analysis;
(c) fragmenting polypeptides derived from the cells into peptide fragments by enzymatic digestion or by non-enzymatic fragmentation;
(d) contacting the labeling reagents of step (b) with the peptide fragments of step (c), thereby labeling the peptides with the differential labeling reagents, wherein the labels used in one same are different from the labels used in other samples;
(e) separating the peptides by chromatography to generate an eluate;
(f) feeding the eluate of step (e) into a mass spectrometer and quantifying the amount of each peptide and generating the sequence of each peptide by use of the mass spectrometer;
(g) inputting the sequence to a computer program product which identifies from which sample each peptide was derived, compares the inputted sequence to a database of polypeptide sequences to identify the polypeptide from which the sequenced peptide originated, and compares the amount of each polypeptide in each sample, thereby quantifying changes in protein expression between at least two cellular states.
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52. A method for identifying proteins by differential labeling of peptides, the method comprising the following steps:
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(a) providing a sample comprising a polypeptide;
(b) providing a plurality of labeling reagents which differ in molecular mass but do not differ in chromatographic retention properties and do not differ in ionization and detection properties in mass spectrographic analysis, wherein the differences in molecular mass are distinguishable by mass spectrographic analysis;
(c) fragmenting the polypeptide into peptide fragments by enzymatic digestion or by non-enzymatic fragmentation;
(d) contacting the labeling reagents of step (b) with the peptide fragments of step (c), thereby labeling the peptides with the differential labeling reagents;
(e) separating the peptides by multidimensional liquid chromatography to generate an eluate;
(f) feeding the eluate of step (e) into a tandem mass spectrometer and quantifying the amount of each peptide and generating the sequence of each peptide by use of the mass spectrometer;
(g) inputting the sequence to a computer program product which compares the inputted sequence to a database of polypeptide sequences to identify the polypeptide from which the sequenced peptide originated.
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53. A chimeric labeling reagent comprising
(a) a first domain comprising a biotin; - and
(b) a second domain comprising a reactive group capable of covalently binding to an amino acid, wherein the chimeric labeling reagent comprises at least one isotope. - View Dependent Claims (54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65)
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66. A method of comparing relative protein concentrations in a sample comprising (a) providing a plurality of differential small molecule tags, wherein the small molecule tags are structurally identical but differ in their isotope composition, and the small molecules comprise reactive groups that covalently bind to cysteine or lysine residues or both;
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(b) providing at least two samples comprising polypeptides;
(c) attaching covalently the differential small molecule tags to amino acids of the polypeptides;
(d) determining the protein concentrations of each sample in a tandem mass spectrometer; and
,(d) comparing relative protein concentrations of each sample. - View Dependent Claims (67, 68, 69, 70, 71)
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72. A method of comparing relative protein concentrations in a sample comprising (a) providing a plurality of differential small molecule tags, wherein the differential small molecule tags comprise a chimeric labeling reagent comprising (i) a first domain comprising a biotin;
- and, (ii) a second domain comprising a reactive group capable of covalently binding to an amino acid, wherein the chimeric labeling reagent comprises at least one isotope;
(b) providing at least two samples comprising polypeptides;
(c) attaching covalently the differential small molecule tags to amino acids of the polypeptides;
(d) isolating the tagged polypeptides on a biotin-binding column by binding tagged polypeptides to the column, washing non-bound materials off the column, and eluting tagged polypeptides off the column;
(e) determining the protein concentrations of each sample in a tandem mass spectrometer; and
,(f) comparing relative protein concentrations of each sample.
- and, (ii) a second domain comprising a reactive group capable of covalently binding to an amino acid, wherein the chimeric labeling reagent comprises at least one isotope;
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73. A method of producing an improved organism having a desirable trait comprising:
- a) obtaining an initial population of organisms, b) generating a set of mutagenized organisms, such that when all the genetic mutations in the set of mutagenized organisms are taken as a whole, there is represented a set of substantial genetic mutations, and c) detecting the presence of said improved organism.
- View Dependent Claims (74, 75, 76, 77, 78, 79, 80, 81, 82, 108, 109)
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83. A method of producing an improved organism having a desirable trait comprising:
- a) obtaining an initial population of organisms, b) generating a set of mutagenized organisms each having at least one genetic mutation, such that when all the genetic mutations in the set of mutagenized organisms are taken as a whole, there is represented a set of substantial genetic mutations c) detecting the manifestation of at least two genetic mutations, d) introducing at least two detected genetic mutations into one organism, and e) optionally repeating any of steps a), b), c), and d).
- View Dependent Claims (84, 85, 86, 87, 88, 89, 90, 91, 92)
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93. A method for identifying a gene that alters a trait of an organism, comprising:
- a) obtaining an initial population of organisms, b) generating a set of mutagenized organisms, such that when all the genetic mutations in the set of mutagenized organisms are taken as a whole, there is represented a set of substantial genetic mutations, and c) detecting the presence an organism having said altered trait, and d) determining the nucleotide sequence of a gene that has been mutagenized in the organism having the altered trait.
- View Dependent Claims (110, 111)
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94. A method for producing an organism with an improved trait, comprising:
- a) functionally knocking out an enogenous gene in a substantially clonal population of organisms;
b) transferring a library of altered genes into the substantially clonal population of organisms, wherein each altered gene differs from the endogenous gene at only one codon;
c) detecting a mutagenized organism having an improved trait; and
d) determining the nucleotide sequence of an gene that has been transferred into the detected organism. - View Dependent Claims (112, 113)
- a) functionally knocking out an enogenous gene in a substantially clonal population of organisms;
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95. A method of introducing differentially activatable stacked traits into a transgenic cell or organism, which method is comprised of the following steps:
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a) obtaining an initial cell or organism;
b) introducing into the working cell or organism a plurality of traits (stacked traits), including selectively and differentially activatable traits, whereby serviceable traits for this purpose include traits conferred by genes and traits conferred by gene pathways;
c) analyzing the information obtained from steps a) and b), and d) optionally repeating any number or all of the steps of a), b), c), and d);
- View Dependent Claims (96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107)
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114. A method for whole cell engineering of new or modified phenotypes by using real-time metabolic flux analysis, the method comprising the following steps:
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(a) making a modified cell by modifying the genetic composition of a cell;
(b) culturing the modified cell to generate a plurality of modified cells;
(c) measuring at least one metabolic parameter of the cell by monitoring the cell culture of step (b) in real time; and
,(d) analyzing the data of step (c) to determine if the measured parameter differs from a comparable measurement in an unmodified cell under similar conditions, thereby identifyng an engineered phenotype in the cell using real-time metabolic flux analysis. - View Dependent Claims (115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179)
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Specification