Mini-Tn7 insertion in bacteria with multiple glmS-linked attTn7 sites:: example Burkholderia mallei ATCC 23344
Mini-Tn7 insertion in bacteria with multiple glmS-linked attTn7 sites:: example Burkholderia mallei ATCC 23344
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DOI：
10.1038/nprot.2006.25
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Schweizer, Herbert P.
中科院分区：
文献类型：
--
作者：
Choi, Kyoung-Hee;DeShazer, David;Schweizer, Herbert P.
迷你 Tn7 载体普遍适用于革兰氏阴性细菌，从而有利于对许多遗传系统很少的生物体进行操作。这些载体，当仅提供 Tn7 位点特异性转座机制时，将位点和方向特异性地插入细菌染色体中必需 glmS 基因下游的 attTn7 位点处。包括伯克霍尔德杆菌在内的一些细菌含有多个 glmS 基因，因此含有多个 attTn7 位点。在这里，我们提供了在鼻疽中应用迷你 Tn7 系统的协议，作为具有多个 glmS 位点的细菌的示例。该过程首先涉及将感兴趣的基因克隆到适当的迷你 Tn7 载体中；其次，通过接合将重组迷你Tn7载体和编码Tn7位点特异性转座途径的辅助质粒共转移到鼻疽杆菌中，然后选择含有插入的菌株；最后，对 mini-Tn7 插入进行 PCR 验证。鼻疽在 1 号染色体上拥有两个 glmS 基因，并且 Tn7 转座到这两个位点，尽管在超过 90% 的检查克隆中发生了与 glmS1 相关的 attTn7-1 转座。转置效率高，从启动到插入事件验证的整个过程可以在不到 5 d 的时间内完成。鼻疽伯克霍尔德氏菌中的第一个染色体整合系统为伯克霍尔德氏菌的遗传工具做出了重要贡献。载体可用于基因互补和表达以及基因融合分析。
The mini-Tn7 vectors are universally applicable in Gram-negative bacteria and thereby facilitate the manipulation of many organisms for which few genetic systems are available. These vectors, when provided with only the Tn7 site-specific transposition machinery, insert site and orientation specifically in the bacterial chromosome at an attTn7 site downstream of the essential glmS gene. A few bacteria, including Burkholderia spp., contain multiple glmS genes and therefore several attTn7 sites. Here we provide a protocol for application of the mini-Tn7 system in B. mallei as an example of bacteria with multiple glmS sites. The procedure involves, first, cloning of the genes of interest into an appropriate mini-Tn7 vector; second, co-transfer of the recombinant mini-Tn7 vector and a helper plasmid encoding the Tn7 site-specific transposition pathway into B. mallei by conjugation, followed by selection of insertion-containing strains; and last, PCR verification of mini-Tn7 insertions. B. mallei possesses two glmS genes on chromosome 1 and Tn7 transposes to both sites, although transposition to attTn7-1 associated with glmS1 occurs in more than 90% of the clones examined. Transposition is efficient and the whole procedure from start to verification of insertion events can be done in less than 5 d. This first chromosome integration system in B. mallei provides an important contribution to the genetic tools emerging for Burkholderia spp. Vectors are available for gene complementation and expression, and gene fusion analyses.