Enhancing cell competence by recursive sequence recombination
AssigneeMaxygen, Inc.
InventorWillem P. C. Stemmer
Life of this patent
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Abstract
The invention provides a number of strategies for transferring and/or evolving gene(s) associated with cellular DNA uptake so that they confer or enhance DNA-uptake capacity of a recipient cell. Evolution is achieved by recursive cycles of recombination and screening/selection. One such strategy entails evolving genes that confer competence in one species to confer either greater competence in that species, or comparable or greater competence in a second species. Another strategy entails evolving genes for use as components of cloning vector to confer enhanced uptake of the vector. Other strategies entail evolving viral receptors, viruses, and genes that mediate conjugal transfer.
Claims33
A method of enhancing competence of a non-mammalian cell, comprising:
- (1) recombining at least first and second DNA segments from at least one gene conferring DNA competence, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes;
- (2) screening the library of recombinant genes for at least one recombinant gene that confers enhanced competence in the non-mammalian cell relative to a wild-type form of the gene;
- (3) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from the first and second segments, to produce a further library of recombinant genes;
- (4) screening the further library of recombinant genes for at least one further recombinant gene that confers enhanced competence in the non-mammalian cell relative to the at least one recombinant gene;
- (5) repeating (3) and (4), as necessary, until the at least one further recombinant gene confers a desired level of enhanced competence in the non-mammalian cell;
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- (6) introducing the at least one further recombinant gene to the non-mammalian cell, thereby conferring enhanced competence.
A recombinant non-mammalian cell which comprises an exogenous gene conferring enhanced competence relative to the wild-type cell, wherein the exogenous gene is from a different species than the non-mammalian cell.
A method of enhancing competence of a non-mammalian cell, comprising:
- (1) conducting a multi-cyclic polynucleotide extension process on annealed polynucleotide strands having sequences from a plurality of initial polynucleotide variants of a gene conferring competence to a cell, the annealed polynucleotide strands having regions of similarity and regions of heterology with each other and being annealed through the regions of similarity, under conditions whereby one strand serves as a template for extension of another strand with which it is annealed to generate a population of recombinant polynucleotides comprising sequences from the different initial variant polynucleotides, which form the annealed polynucleotide strands for a subsequent extension cycle;
- (2) selecting or screening the recombinant polynucleotides to identify a recombinant polynucleotide that confers enhanced competence to a non-mammalian cell;
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- (3) introducing into the non-mammalian cell the recombinant polynucleotide identified in step (2), thereby conferring enhanced competence to the cell.
Dependencies are inferred from the claim text.