A broad-host-range F1p-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: Application for isolation of unmarked Pseudomonas aeruginosa mutants

Tung T. Hoang, Roxann R. Karkhoff-Schweizer, Alecksandr J. Kutchma, Herbert P. Schweizer

Research output: Contribution to journalArticlepeer-review

1470 Scopus citations

Abstract

An improved method for gene replacement in Pseudomonas aeruginosa was developed. The method employs several new gene replacement vectors that incorporate (1) the counterselectable sacB marker, (2) a lacZα-allele for blue-white screening, (3) the pUC18/19 vectors multiple cloning site with 10 unique restriction sites, (4) an oriT for conjugation-mediated plasmid transfer and (5) carbenicillin, gentamicin (Gm) and tetracycline selectable markers. A cassette was constructed that contains a Gm(R) selectable marker next to the green fluorescent protein structural gene, with both markers being flanked by F1p recombinase target (FRT) sites. The FRT cassette was used to insertionally inactivate the cloned P. aeruginosa pabC gene encoding aminodeoxychorismate lyase. After conjugal transfer into P. aeruginosa, plasmid integrants were selected, and deletion of unwanted DNA sequences was promoted by sucrose counterselection. The FRT cassette was excised with high frequencies (close to 100%) from the chromosome after conjugal transfer of a F1p recombinase-expressing plasmid; this sacB-containing plasmid was subsequently cured by sucrose counterselection, resulting in an unmarked P. aeruginosa ΔpabC strain.

Original languageEnglish (US)
Pages (from-to)77-86
Number of pages10
JournalGene
Volume212
Issue number1
DOIs
StatePublished - May 28 1998
Externally publishedYes

Keywords

  • Cassette
  • Gene replacement
  • Mutagenesis
  • Saccharomyces cerevisiae

ASJC Scopus subject areas

  • Genetics

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