Methods of making unbiased phage libraries

A method of preparing an unbiased mutational phage library of a target phage gene, comprising

• preparing a population of acceptor phages having a genome comprising recombination sites by providing a base population of lytic phages having a lytic phage genome, removing an endogenous target phage gene from the lytic phage genome, and inserting the recombination sites into the lytic phage genome;
• recombining a mutated target phage gene into a portion of the population of acceptor phages by replicating the population of acceptor phages within a first host bacteria, the first host bacteria comprising a first helper plasmid and a recombination plasmid, wherein the first helper plasmid expresses the endogenous target phage gene, and the recombination plasmid comprises the mutated target phage gene and complimentary recombination sites and expresses a recombinase, wherein the recombining produces a mixed phage population comprising acceptor phages and recombined phages, wherein the recombined phages comprise the mutated target phage gene in their genomes;
• selectively accumulating the recombined phages from the mixed phage population by replicating the mixed phage population within a second host bacteria, the second host bacteria comprising a second helper plasmid and a counterselection system, wherein the second helper plasmid expresses the endogenous target phage gene, wherein the counterselection system suppresses replication of the acceptor phages, and wherein replication of acceptor phages is suppressed and replication of recombined phages is amplified to provide a population of accumulated recombined phages;
• and
• replicating the population of accumulated recombined phage within a third host bacteria, without a helper plasmid, to provide the unbiased phage library,
• wherein the population of lytic phages lyses the first, second and third bacteria.