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

Gene
T T HoangH P Schweizer

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 lacZ alpha-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 GmR selectable marker next to the green fluorescent protein structural gene, with both markers being flanked by Flp 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 Flp recombinase-expressing plasmid; this sacB-containing plasmid was subsequently cured by sucrose counterselection, resulting in an unmarked P. aeruginosa delta pabC strain.

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Citations

Jul 28, 2004·Archives of Microbiology·Dayna M BrichtaGerard A O'Donovan
Jun 25, 2008·Archives of Microbiology·Utta KretzschmarHelmut Görisch
Nov 11, 2009·Archives of Microbiology·Nicole GlieseHelmut Görisch
Jun 16, 2010·Archives of Microbiology·Utta KretzschmarLorenz Adrian
May 29, 2010·Applied Microbiology and Biotechnology·Xiao-Xing WeiGuo-Qiang Chen
Sep 22, 2010·Applied Microbiology and Biotechnology·Ling HuangXue-Hong Zhang
Oct 20, 2010·Applied Microbiology and Biotechnology·Aidan L KerrBrett A Neilan
Oct 11, 2011·Applied Microbiology and Biotechnology·Ben RaoYaling Shen
Feb 9, 2013·Applied Microbiology and Biotechnology·Paramasivan PonrajParamasamy Gunasekaran
Apr 18, 2013·Current Microbiology·Carl J BalibarCharles R Dean
Jul 19, 2011·Journal of Bioenergetics and Biomembranes·Toshihede MatsunoIsao Yumoto
May 18, 2004·Journal of the American Society for Mass Spectrometry·François LépineLaurence G Rahme
Mar 13, 2012·Journal of Microbiological Methods·Jerónimo Rodríguez-BeltránAlexandro Rodríguez-Rojas
Oct 18, 2001·Current Opinion in Biotechnology·H P Schweizer
May 24, 2005·Nature Methods·Kyoung-Hee ChoiHerbert P Schweizer
Apr 5, 2007·Nature Protocols·Kyoung-Hee Choi, Herbert P Schweizer
Nov 8, 2002·Molecular Microbiology·Michelle L McCawTimothy L Yahr
Jan 22, 2003·Environmental Microbiology·Craig DanielsJoseph S Lam

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