We present a scarless recombineering-based method for introducing multiple point mutations into the genome of a temperate phage. The method uses the λ Red recombineering system to promote exogenous ssDNA oligos to anneal on the prophage lagging strand during host genome replication. DNA repair is suppressed by inducing the expression of a dominant-negative mutant protein of the methyl-directed mismatch repair system. Screening for recombinant cells without a selection marker is feasible due to its high recombination frequency, estimated as more than 40% after six cycles. The method enables scarless editing of the genome of a bacteriophage in 4-5 days.
Keywords: Bacteriophage; Electroporation; Lysogenic; Recombineering; Scarless; Temperate; ssDNA; λ Red.
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