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US 6,528,311 B1Granted
Evolution of whole cells and organisms by recursive sequence recombination
AssigneeMaxygen, Inc.
InventorsStephen delCardayre, Matthew Tobin, William P. C. Stemmer, Jon E. Ness, Jeremy Minshull, Phillip Patten, Venkiteswaran Subramanian, Linda Castle, Claus M. Krebber, Steve Bass
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Abstract
The invention provides methods employing iterative cycles of recombination and selection/screening for evolution of whole cells and organisms toward acquisition of desired properties. Examples of such properties include enhanced recombinogenicity, genome copy number, and capacity for expression and/or secretion of proteins and secondary metabolites.
Claims64
A method of generating a cell with a desired property, the method comprising:
- (a) providing a set of parental cells, comprising at least 3 different parental members, which set of parental cells comprises a plurality of variant polynucleotides, wherein the plurality of variant polynucleotides differ from each other by at least two nucleotides;
- (b) propagating the parental cells under conditions whereby at least one round of nucleic acid exchange occurs between at least 3 parental cells, providing for recombination of the plurality of polynucleotide variants in the cells, thereby generating a plurality of recombinant nucleic acids within a plurality of resulting recombinant cells, each of which recombinant cells comprises nucleic acids derived from the at least 3 different parental cells;
- (c) propagating the recombinant cells under conditions whereby at least one round of nucleic acid exchange occurs between a plurality of the recombinant cells, providing for recombination of the plurality of recombinant nucleic acids in the recombinant cells, thereby generating a plurality of additionally recombinant nucleic acids within a plurality of resulting progeny cells;
- and,
- (d) identifying at least one progeny cell comprising a recombinant nucleic acid that confers the desired property.
2 more elements
A method of generating a cell with a desired property, the method comprising:
- (a) providing a set of parental cells comprising at least 3 different parental members, which set of parental cells comprises a plurality of variant polynucleotides, wherein the plurality of variant polynucleotides differ from each other by at least two nucleotides;
- (b) propagating the parental cells under conditions whereby at least one round of nucleic acid exchange occurs between at least 3 different parental cells, wherein the nucleic acid exchange is mediated by transducing phage particles, thereby forming transduced cells, and wherein the exchanged nucleic acid comprises cellular genomic DNA, providing for recombination of the plurality of polynucleotide variants, and thereby generating a plurality of recombinant nucleic acids within a plurality of resulting recombinant cells, each of which recombinant cells comprises nucleic acids derived from the at least 3 different parental cells;
- and,
- (c) identifying at least one progeny cell comprising a recombinant nucleic acid that confers the desired property.
1 more element
A method of generating a bacterial cell with a desired property, the method comprising:
- (a) providing a set of bacterial cells comprising a plurality of variant genomic polynucleotides, wherein the plurality of variant genomic polynucleotides differ from each other by at least two nucleotides;
- (b) providing a plurality of transducing phage particles comprising one or more members of the plurality of variant genomic polynucleotides;
- (c) infecting the bacterial cells with the transducing phage particles under conditions whereby recombination of two or more members of the plurality of variant genomic polynucleotide occurs in one or more transduced bacterial cells, thereby generating a plurality of recombinant nucleic acids within a plurality of resulting recombinant bacterial cells;
- (d) propagating the plurality of resulting recombinant bacterial cells under conditions whereby at least one additional round of nucleic acid exchange occurs between the recombinant bacterial cells, providing for further recombination of the plurality of recombinant nucleic acids, and thereby generating a plurality of additionally recombinant nucleic acids within a plurality of further recombinant bacterial cells;
- and,
- (e) identifying at least one progeny cell comprising a recombinant nucleic acid that confers the desired property.
3 more elements
Dependencies are inferred from the claim text.