Rapid and highly efficient method for scarless mutagenesis within the Salmonella enterica chromosome.
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-SceI endonuclease-based selection of successful recombinants after electroporation with short synthetic oligonucleotides. 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.
Junior Research Group 3, Robert Koch Institute, Wernigerode Branch, Wernigerode, Germany.
Kathrin
K
Blank
Michael
M
Hensel
Roman G
RG
Gerlach
D002876:Chromosomes, Bacterial / Q000235:genetics*; D008722:Methods; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D017354:Point Mutation; D011995:Recombination, Genetic; D019779:Salmonella enterica / Q000235:genetics*; D017384:Sequence Deletion
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