TY - JOUR
T1 - A broad-host-range F1p-FRT recombination system for site-specific excision of chromosomally-located DNA sequences
T2 - Application for isolation of unmarked Pseudomonas aeruginosa mutants
AU - Hoang, Tung T.
AU - Karkhoff-Schweizer, Roxann R.
AU - Kutchma, Alecksandr J.
AU - Schweizer, Herbert P.
N1 - Funding Information:
This work was supported by a grant from the Research Council of the College of Veterinary Medicine and Biomedical Sciences at Colorado State University and, in part, by start up funds from the Department of Microbiology. We wish to thank Dr W. Wackernagel, University of Oldenburg, and Dr M.L. Vasil, University of Colorado Health Sciences Center, for the provision of bacterial plasmids.
PY - 1998/5/28
Y1 - 1998/5/28
N2 - 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.
AB - 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.
KW - Cassette
KW - Gene replacement
KW - Mutagenesis
KW - Saccharomyces cerevisiae
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U2 - 10.1016/S0378-1119(98)00130-9
DO - 10.1016/S0378-1119(98)00130-9
M3 - Article
C2 - 9661666
AN - SCOPUS:0032575051
SN - 0378-1119
VL - 212
SP - 77
EP - 86
JO - Gene
JF - Gene
IS - 1
ER -