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

Herbert P. Schweizer, Tung T. Hoang

Research output: Contribution to journalArticlepeer-review

314 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::GmR fusion cassette into the glpD gene of Pseudomonas aeruginosa.

Original languageEnglish (US)
Pages (from-to)15-22
Number of pages8
JournalGene
Volume158
Issue number1
DOIs
StatePublished - 1995
Externally publishedYes

Keywords

  • Blue-white screening
  • conjugation
  • gentamycin resistance
  • sacB
  • xylE cassette

ASJC Scopus subject areas

  • Genetics

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