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Applied and Environmental Microbiology, March 2009, p. 1575-1580, Vol. 75, No. 6
0099-2240/09/$08.00+0     doi:10.1128/AEM.02509-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Rapid Oligonucleotide-Based Recombineering of the Chromosome of Salmonella enterica{triangledown} ,{dagger}

Roman G. Gerlach,{ddagger} Daniela Jäckel, Stefanie U. Hölzer, and Michael Hensel*

Infektionsbiologische Abteilung, Universitätsklinikum Erlangen, Erlangen, Germany

Received 2 November 2008/ Accepted 6 January 2009

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.

* Corresponding author. Mailing address: Infektionsbiologische Abteilung im Mikrobiologischen Institut, Universitätsklinikum Erlangen, Wasserturmstr. 3-5, D-91054 Erlangen, Germany. Phone: 49 (0)9131 85 23640. Fax: 49 (0)9131 85 22531. E-mail: Michael.Hensel{at}uk-erlangen.de

{triangledown} Published ahead of print on 16 January 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{ddagger} Present address: Robert Koch-Institut, Wernigerode Branch, Burgstr. 37, D-38855 Wernigerode, Germany.

Applied and Environmental Microbiology, March 2009, p. 1575-1580, Vol. 75, No. 6
0099-2240/09/$08.00+0     doi:10.1128/AEM.02509-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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