An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants.

ABSTRACT:

Background

Traditional gene replacement procedures are still time-consuming. They usually necessitate cloning of the gene to be mutated, insertional inactivation of the gene with an antibiotic resistance cassette and exchange of the plasmid-borne mutant allele with the bacterial chromosome. PCR and recombinational technologies can be exploited to substantially accelerate virtually all steps involved in the gene replacement process.

Results

We describe a method for rapid generation of unmarked P. aeruginosa deletion mutants. Three partially overlapping DNA fragments are amplified and then spliced together in vitro by overlap extension PCR. The resulting DNA fragment is cloned in vitro into the Gateway vector pDONR221 and then recombined into the Gateway-compatible gene replac

SUBMITTER: Choi KH 

PROVIDER: S-EPMC1173109 | biostudies-literature | 2005 May

REPOSITORIES: biostudies-literature