Evolution of site-specific recombinases

A method for evolving a recombinase, the method comprising (a) contacting a population of host cells with a population of phage vectors comprising a gene encoding a recombinase and deficient in at least one gene for the generation of infectious phage particles, wherein (1) the host cells are amenable to transfer of the phage vector;

• (2) the vector allows for expression of the recombinase in a host cell, can be replicated by the host cell, and the replicated vector can transfer from host cell to host cell;
• (3) the host cells express a gene product encoded by the at least one gene for the generation of infectious phage particles of (a) in response to the recombination of a recombinase target sequence by the recombinase, and the level of gene product expression depends on the activity of the recombinase towards the target sequence;
• (b) incubating the population of host cells under conditions allowing for mutation of the gene encoding the recombinase and the transfer of the phage vectors from host cell to host cell, wherein host cells are removed from the host cell population, and the population of host cells is replenished with fresh host cells that do not harbor the phage vector;
• (c) isolating a replicated phage vector from the host cell population in (b), wherein the replicated vector comprises a mutated version of the gene encoding the recombinase,
• wherein the host cells harbor an expression construct comprising a nucleotide sequence encoding a gene product of the at least one gene for the generation of infectious phage particles of (a) under the control of a heterologous promoter and a transcriptional terminator flanked by two recombinase target sequences, wherein the recombinase target sequences are different from the target sequences recognized by the wild-type version of the recombinase, wherein recombination of the recombinase target sequences results in excision of the transcriptional terminator and expression of the at least one gene for the generation of infectious phage particles,
• wherein the phage vector is a filamentous phage, wherein the phage vector is an M13 phage, an fl phage, or an fd phage, wherein the at least one gene for the generation of infectious phage particles comprises a sequence encoding a pIII protein.