Multiple-site genetic modifications in Escherichia coli using lambda-Red recombination and I-SceI cleavage

OBJECTIVES: Genetic modifications to bacterial chromosomes are important for research; recently we reported a two-plasmid system for single locus modification in Escherichia coli and an improved method for simultaneous multiple-loci modification is needed. RESULTS: An intermediate bacterial strain was generated with different resistance marker genes flanked by I-SceI recognition sites at multiple target loci. Then a donor plasmid carrying several alleles with desired modifications was transformed into the intermediate strain together with a bifunctional helper plasmid encoding λ-Red recombinase and I-SceI endonuclease. I-SceI would induce double-strand breaks (DSBs) in the chromosome and λ-Red would induce recombination between chromosome DSBs and allele fragments from the donor plasmid, resulting in genomic modifications. CONCLUSIONS: This method has been used to successfully perform three different loci modifications simultaneously.