Scarless and sequential gene modification in Pseudomonas using PCR product flanked by short homology regions.

ABSTRACT:

Background

The lambda Red recombination system has been used to inactivate chromosomal genes in various bacteria and fungi. The procedure consists of electroporating a polymerase chain reaction (PCR) fragment containing antibiotic cassette flanked by homology regions to the target locus into a strain that can express the lambda Red proteins (Gam, Bet, Exo).

Results

Here a scarless gene modification strategy based on the Red recombination system has been developed to modify Pseudomonas genome DNA via sequential deletion of multiple targets. This process was mediated by plasmid pRKaraRed encoding the Red proteins regulated by PBAD promoter, which was functional in P. aeruginosa as well as in other bacteria. First the target gene was substituted for the sacB-bla cassette flanke

SUBMITTER: Liang R 

PROVIDER: S-EPMC2924854 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature