An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa

Herbert P. Schweizer; Tung T. Hoang

doi:10.1016/0378-1119(95)00055-B

An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa

Herbert P. Schweizer, Tung T. Hoang

Research output: Contribution to journal › Article › peer-review

321 Scopus citations

Abstract

A novel pUC19-based gene replacement vector has been developed. This vector incorporates (i) the counterselectable sacB marker, (ii) a lacZα allele for blue-white screening, (iii) an oriT for conjugation-mediated plasmid transfer and (iv) unique cloning sites for SmaI and the rare-cutting meganuclease I-SceI. These rare restriction sites are also present on the helper plasmid pUC19Sce. The replacement vector is engineered to contain few restriction sites to gain greater access to restriction sites within cloned DNA fragments, thus facilitating their genetic manipulation. The usefulness of the system was demonstrated by chromosomal integration of a newly constructed xylE::Gm^R fusion cassette into the glpD gene of Pseudomonas aeruginosa.

Original language	English (US)
Pages (from-to)	15-22
Number of pages	8
Journal	Gene
Volume	158
Issue number	1
DOIs	https://doi.org/10.1016/0378-1119(95)00055-B
State	Published - 1995
Externally published	Yes

Keywords

Blue-white screening
conjugation
gentamycin resistance
sacB
xylE cassette

ASJC Scopus subject areas

Genetics

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10.1016/0378-1119(95)00055-B

Cite this

@article{f8cd6fcc04874abeabb97c532c19e0ba,

title = "An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa",

abstract = "A novel pUC19-based gene replacement vector has been developed. This vector incorporates (i) the counterselectable sacB marker, (ii) a lacZα allele for blue-white screening, (iii) an oriT for conjugation-mediated plasmid transfer and (iv) unique cloning sites for SmaI and the rare-cutting meganuclease I-SceI. These rare restriction sites are also present on the helper plasmid pUC19Sce. The replacement vector is engineered to contain few restriction sites to gain greater access to restriction sites within cloned DNA fragments, thus facilitating their genetic manipulation. The usefulness of the system was demonstrated by chromosomal integration of a newly constructed xylE::GmR fusion cassette into the glpD gene of Pseudomonas aeruginosa.",

keywords = "Blue-white screening, conjugation, gentamycin resistance, sacB, xylE cassette",

author = "Schweizer, {Herbert P.} and Hoang, {Tung T.}",

note = "Funding Information: This work wass upport,~bdy grantsfr omt heCanadian Cystic Fibrosis Foundationa nd the Medical Research Council of Canada (MT-11245). H.P.S. is a MRC Medical Scholar.W e thank M. Bacic for pPS333 and assistancew ith the C230 assays,a nd T. de Kievit, Universityo f Guelph,f or communicatinugn published results.",

year = "1995",

doi = "10.1016/0378-1119(95)00055-B",

language = "English (US)",

volume = "158",

pages = "15--22",

journal = "Gene",

issn = "0378-1119",

publisher = "Elsevier BV",

number = "1",

}

TY - JOUR

T1 - An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa

AU - Schweizer, Herbert P.

AU - Hoang, Tung T.

N1 - Funding Information: This work wass upport,~bdy grantsfr omt heCanadian Cystic Fibrosis Foundationa nd the Medical Research Council of Canada (MT-11245). H.P.S. is a MRC Medical Scholar.W e thank M. Bacic for pPS333 and assistancew ith the C230 assays,a nd T. de Kievit, Universityo f Guelph,f or communicatinugn published results.

PY - 1995

Y1 - 1995

N2 - A novel pUC19-based gene replacement vector has been developed. This vector incorporates (i) the counterselectable sacB marker, (ii) a lacZα allele for blue-white screening, (iii) an oriT for conjugation-mediated plasmid transfer and (iv) unique cloning sites for SmaI and the rare-cutting meganuclease I-SceI. These rare restriction sites are also present on the helper plasmid pUC19Sce. The replacement vector is engineered to contain few restriction sites to gain greater access to restriction sites within cloned DNA fragments, thus facilitating their genetic manipulation. The usefulness of the system was demonstrated by chromosomal integration of a newly constructed xylE::GmR fusion cassette into the glpD gene of Pseudomonas aeruginosa.

AB - A novel pUC19-based gene replacement vector has been developed. This vector incorporates (i) the counterselectable sacB marker, (ii) a lacZα allele for blue-white screening, (iii) an oriT for conjugation-mediated plasmid transfer and (iv) unique cloning sites for SmaI and the rare-cutting meganuclease I-SceI. These rare restriction sites are also present on the helper plasmid pUC19Sce. The replacement vector is engineered to contain few restriction sites to gain greater access to restriction sites within cloned DNA fragments, thus facilitating their genetic manipulation. The usefulness of the system was demonstrated by chromosomal integration of a newly constructed xylE::GmR fusion cassette into the glpD gene of Pseudomonas aeruginosa.

KW - Blue-white screening

KW - conjugation

KW - gentamycin resistance

KW - sacB

KW - xylE cassette

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UR - http://www.scopus.com/inward/citedby.url?scp=0029061955&partnerID=8YFLogxK

U2 - 10.1016/0378-1119(95)00055-B

DO - 10.1016/0378-1119(95)00055-B

M3 - Article

C2 - 7789804

SN - 0378-1119

VL - 158

SP - 15

EP - 22

JO - Gene

JF - Gene

IS - 1

ER -

An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa

Abstract

Keywords

ASJC Scopus subject areas

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