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Direct manipulation of bacterial chromosomes by recombination-based techniques has become increasingly important for both cognitive and applied research. Here we demonstrate, for the first time, the combination of the Red recombinase system with I- Sce I endonuclease-based selection of successful recombinants after electroporation with short synthetic olignucleotides. We show the generation of scarless gene knockouts as well as site-directed mutagenesis using the  Salmonella  virulence-associated two component signaling system PhoPQ. The presented approach is very versatile for generating in-frame deletions, point mutations or insertions within bacterial chromosomes.

Rapid and Highly Efficient Method for Scarless Mutagenesis within the Salmonella enterica Chromosome