Rapid oligonucleotide-based recombineering of the chromosome of Salmonella enterica.
Recombinant engineering using Red recombinase-based approaches offers efficient and rapid approaches to deletion and modification of genes. Here we describe a novel application of Red recombinant engineering that enables direct manipulation of chromosomal loci by electroporation with short synthetic DNA molecules. We demonstrate the use of this approach for the generation of scarless in-frame deletions in chromosomal genes of Salmonella enterica. Furthermore, we describe rapid site-directed mutagenesis within bacterial chromosomes without any requirement for cloning and mutating genes in vitro or for reintroducing mutant alleles into the chromosome. This approach can be expected to facilitate mutational analysis in S. enterica and in other bacterial species able to support Red-mediated recombination.
Gerlach
Roman G
RG
Infektionsbiologische Abteilung im Mikrobiologischen Institut, Universitätsklinikum Erlangen, Wasserturmstr. 3-5, D-91054 Erlangen, Germany.
Jäckel
Daniela
D
Hölzer
Stefanie U
SU
Hensel
Michael
M
D002876:Chromosomes, Bacterial; D018274:Electroporation; D017353:Gene Deletion; D005827:Genetics, Microbial / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000235:genetics; D011995:Recombination, Genetic; D019779:Salmonella enterica / Q000235:genetics*; D014169:Transformation, Bacterial
D009841:Oligonucleotides