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
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
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DOI：
10.1016/s0167-7012(01)00246-9
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发表时间：
2001-07-01
影响因子：
2.2
通讯作者：
Nybroe, O
中科院分区：
文献类型：
--
作者：
Koch, B;Jensen, LE;Nybroe, O
由于缺乏合适的载体，使用基于Tn 7的系统将DNA位点特异性插入革兰氏阴性细菌的染色体中受到限制。因此，我们开发了一组灵活的Tn 7递送载体。在一组载体中，插入染色体中的miniTn 7元件含有多克隆位点（MCS）和卡那霉素、链霉素或庆大霉素抗性标记。另一组用于标记绿色荧光蛋白（GFP）的载体携带由修饰的lac启动子P-A1/04/03控制的gfpmut 3 * 基因、几种转录终止子和各种抗性标记。这些载体将Tn 7插入到测试的荧光假单胞菌菌株中编码葡糖胺-6-磷酸合成酶（GlmS）的基因的紧下游的特异性中性基因间区域中。含有庆大霉素抗性标记的GFP标记载体可用于标记携带Tn 5转座子的菌株。Tn 5转座子通常携带卡那霉素抗性编码基因，并且经常用于产生细菌突变体和在基因表达研究中递送报告子构建体。为了证明双标记/报告系统的实用性，在荧光假单胞菌中将Tn 7-gfp标记系统与Tn 5递送的luxAB报告系统组合。该系统允许检测GFP标记的细胞在大麦根际，而Tn 5标记的基因座的表达可以通过测量生物发光来确定。(C)2001年爱思唯尔科学B. V.所有晚上保留。
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.