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.
Journal article
2022-01-01T00:00:00+00:00
2479
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Bacteriophage, Electroporation, Lysogenic, Recombineering, Scarless, Temperate, ssDNA, λ Red, Bacteriophage lambda, DNA, Single-Stranded, Genetic Engineering, Lysogeny, Point Mutation, Prophages