A Tn7-based broad-range bacterial cloning and expression system
A Tn7-based broad-range bacterial cloning and expression system
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DOI：
10.1038/nmeth765
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发表时间：
2005-06-01
期刊：
影响因子：
48
通讯作者：
Schweizer, HP
中科院分区：
文献类型：
--
作者：
Choi, KH;Gaynor, JB;Schweizer, HP
对于许多细菌而言，克隆和表达系统要么稀缺，要么不存在。我们构建了几种迷你Tn7载体，并评估了它们作为广泛适用的克隆和表达系统的潜力。在具有单条染色体的细菌中，包括铜绿假单胞菌、恶臭假单胞菌和鼠疫耶尔森菌，并且在存在编码位点特异性转座途径的辅助质粒的情况下，位点和方向特异性的Tn7插入发生在glmS基因下游的单个attTn7位点。泰国伯克霍尔德菌包含两条染色体，每条染色体都含有一个glmS基因和一个attTn7位点。Tn7系统允许将多种遗传性状导入细菌，正如通过弥补铜绿假单胞菌生物膜生长缺陷、在铜绿假单胞菌和恶臭假单胞菌中建立表达系统以及对鼠疫耶尔森菌进行“绿色荧光蛋白标记”所证明的那样。因此，该系统将在生物医学和环境领域有广泛应用，特别是在质粒和抗生素筛选不可行的环境中，即在植物和动物模型或生物膜中。
For many bacteria, cloning and expression systems are either scarce or nonexistent. We constructed several mini-Tn7 vectors and evaluated their potential as broad-range cloning and expression systems. In bacteria with a single chromosome, including Pseudomonas aeruginosa, Pseudomonas putida and Yersinia pestis, and in the presence of a helper plasmid encoding the site-specific transposition pathway, site- and orientation-specific Tn7 insertions occurred at a single attTn7 site downstream of the glmS gene. Burkholderia thailandensis contains two chromosomes, each containing a glmS gene and an attTn7 site. The Tn7 system allows engineering of diverse genetic traits into bacteria, as demonstrated by complementing a biofilm-growth defect of P. aeruginoso, establishing expression systems in P. aeruginosa and P. putida, and 'GFP-tagging' Y. pestis. This system will thus have widespread biomedical and environmental applications, especially in environments where plasmids and antibiotic selection are not feasible, namely in plant and animal models or biofilms.