Evolution of whole cells and organisms by recursive sequence recombination

A method of performing in vivo recombination, the method of comprising:

• providing a cell incapable of expressing a cell septation gene;
• introducing at least first and second segments from at least one gene into a cell, the segments differing from each other in at least two nucleotides, whereby the segments recombine to produce a library of chimeric genes;
• and
• selecting a chimeric gene from the library having an acquired function.

A method of predicting efficacy of a drug in treating a viral infection, the method comprising:

• (i) recombining a nucleic acid segment from a virus, whose infection is inhibited by a drug, with at least a second nucleic acid segment from the virus, the second nucleic acid segment differing from the nucleic acid segment in at least two nucleotides, to produce a library of recombinant nucleic acid segments;
• (ii) contacting host cells with a collection of viruses having genomes including the recombinant nucleic acid segments in a media containing the drug, and collecting progeny viruses resulting from infection of the host cells,
• (iii) recombining a recombinant DNA segment from a first progeny virus with at least a recombinant DNA segment from a second progeny virus to produce a further library of recombinant nucleic acid segments;
• (iv) contacting host cells with a collection of viruses having genomes including the further library or recombinant nucleic acid segments, in media containing the drug, and collecting further progeny viruses produced by the host cells, and,
• (v) repeating (iii) and (iv), as necessary, until a further progeny virus has acquired a desired degree of resistance to the drug, whereby the degree of resistance acquired and the number of repetitions of (iii) and (iv) needed to acquire it provide a measure of the efficacy of the drug in treating the virus.

A method of evolving a cell to acquire a desired function, the method comprising:

• (a) providing a population of different cells;
• (b) culturing the cells under conditions whereby DNA is exchanged between cells, forming cells with hybrid genomes;
• (c) screening or selecting the cells for cells that have evolved toward acquisition of a desired property;
• and,
• (d) repeating steps (b) and (c) with the selected or screened cells forming the population of different cells until a cell has acquired the desired property.

A method of evolving a cell toward acquisition of a desired property comprising:

• (a) providing a population of different cells;
• (b) isolating DNA from a first subpopulation of the different cells and encapsulating the DNA in liposomes;
• (c) forming protoplasts from a second subpopulation of the different cells;
• (d) fusing the liposomes with the protoplasts whereby DNA from the liposomes is taken up by the protoplasts and recombines with the genomes of the protoplasts;
• (e) incubating the protoplasts under regenerating conditions;
• (f) selecting or screening for regenerating or regenerated cells that have evolved toward the desired property;
• and,
• (g) repeating steps (a)-(f) with the cells that have evolved toward the desired property forming the population of different cells in step (a).

A method of evolving a DNA segment for acquisition of a desired property, the method comprising:

• (a) providing a library of variants of the segment, each variant cloned into separate copies of an artificial chromosome;
• (b) introducing the copies of the artificial chromosome into a population of cells;
• (c) culturing the cells under conditions whereby sexual recombination occurs between cells and homologous recombination occurs between copies of the artificial chromosome bearing the variants;
• and,
• (d) screening or selecting for variants that have evolved toward acquisition of the desired property.

A method of poolwise recombination and selection of cells, the method comprising:

• (i) introducing a library of DNA fragments into a plurality of recipient cells, whereby a plurality of fragments of the library undergo recombination with a genome or an episome of a plurality of recipient cells to produce a plurality of modified cells, whereby the plurality of modified cells comprise a plurality of different modified fragments;
• (ii) fusing at least two of the modified cells, whereby genomes of the at least two modified cells recombine;
• (iii) segregating the resulting fused cell to produce a plurality of regenerated cells, each with a single genome;
• and,
• (iv) selecting the regenerated cells for a desired property.

A method of increasing resistance to virus infection in cells comprising:

• introducing a shuffled library comprising at least one shuffled interferon gene into animal cells, thereby creating an initial library of animal cells or non-human animals;
• challenging the initial library with the virus;
• selecting animal cells or non-human animals from the initial library which are resistant to the virus;
• recovering a plurality of transgenes from a plurality of animal cells or non-human animals which are resistant to the virus;
• shuffling the plurality of transgenes to produce an evolved library of animal cells or non-human animals;
• challenging the evolved library of animal cells or non-human animals with the virus;
• and,
• selecting cells or non-human animals from the evolved library the which are resistant to the virus.

A method for whole genome shuffling through organized heteroduplex shuffling, the method comprising:

• (a) providing chromosomal DNA of an organism which is targeted for shuffling, digesting the chromosomal DNA with one or more restriction enzymes, ligating the chromosomal DNA into a cosmid, the cosmid comprising at least two rare restriction enzyme recognition sites, aliquoting, purifying, and storing sufficient cosmids to represent a complete chromosome;
• (b) mutagenizing aliquots of the library in vitro using a mutagen;
• (c) transfecting a sample from a plurality of the mutagenized aliquots into a population of target cells;
• (d) assaying resulting transfectants for phenotypic improvements;
• (e) growing transfected cells harboring a mutant library of the identified cosmid(s) on media and screening the resulting cell colonies for independent mutants conferring a desired phenotype;
• (f) isolating and pooling DNA from cells identified in the screening;
• (g) dividing the selected pools and digesting at least one sample with a rare-cutting restriction enzyme, pooling the cleaved samples, denaturing the samples, reannealing the samples and religating the samples;
• and,
• (h) transfecting target cells with the resulting heteroduplexes and propagating the cells to allow recombination to occur between the strands of the heteroduplexes in vivo.

A method of selecting for increased copy number of a nucleic acid sequence contributing to a desired phenotype of a target cell comprising:

• providing a genomic library in a suicide vector comprising a dose-sensitive selectable marker;
• transducing the genomic library into a population of target cells;
• selecting the population of target cells for increasing doses of the selectable marker under conditions in which the suicide vector does not replicate episomally;
• selecting a plurality of target cells for the desired phenotype;
• recombining the selected plurality of target cells;
• and, selecting the selected plurality of target cells for the desired phenotype.