A panel of Tn7-based vectors for insertion of the gfp marker gene or for delivery of cloned DNA into Gram-negative bacteria at a neutral chromosomal site

被引:213
作者
Koch, B [1 ]
Jensen, LE [1 ]
Nybroe, O [1 ]
机构
[1] Royal Vet & Agr Univ, Dept Ecol, Sect Genet & Microbiol, DK-1871 Copenhagen C, Denmark
关键词
GFP; transposon mutagenesis; glmS; Pseudomonas;
D O I
10.1016/S0167-7012(01)00246-9
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
The use of Tn7-based systems for site-specific insertion of DNA into the chromosome of Gram-negative bacteria has been limited due to the lack of appropriate vectors. We therefore developed a flexible panel of Tn7 delivery vectors. In one group of vectors, the miniTn7 element, which is inserted into the chromosome, contains a multiple cloning site (MCS) and the kanamycin, streptomycin or gentamicin resistance markers. Another group of Vectors intended for tagging with green fluorescent protein (GFP) carries the gfpmut3* gene controlled by the modified lac promoter P-A1/04/03, several transcriptional terminators, and various resistance markers. These vectors insert Tn7 into a specific, neutral intergenic region immediately downstream of the gene encoding glucosamine-6-phosphate synthetase (GlmS) in the tested fluorescent Pseudomonas strains. The gfp-tagging vector containing a gentamicin-resistance marker is useful for tagging strains carrying a Tn5 transposon. Tn5 transposons often carry kanamycin-resistance-encoding genes and are frequently used to generate bacterial mutants and to deliver reporter constructions in gene expression studies. To demonstrate the utility of a dual marker / reporter system, the Tn7-gfp marker system was combined with a Tn5-delivered luxAB reporter system in Pseudomonas fluorescens. The system allowed detection of gfp-tagged cells in the barley rhizosphere, while expression of the Tn5-tagged locus could be determined by measuring bioluminescence. (C) 2001 Elsevier Science B.V. All nights reserved.
引用
收藏
页码:187 / 195
页数:9
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