An Improved Method for oriT-Directed Cloning and Functionalization of Large Bacterial Genomic Regions
An Improved Method for oriT-Directed Cloning and Functionalization of Large Bacterial Genomic Regions
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DOI：
10.1128/aem.00994-13
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发表时间：
2013-08-01
影响因子：
4.4
通讯作者：
Schweizer, Herbert P.
中科院分区：
文献类型：
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作者：
Kvitko, Brian H.;McMillan, Ian A.;Schweizer, Herbert P.
我们对广泛宿主范围的系统进行了重大改进，用于基于接合过程中直接重复的 oriT 位点之间的位点特异性重组来克隆和操作大型细菌基因组区域。使用两个携带侧翼同源区域的自杀捕获载体，oriT 位点在目标区域的两侧进行重组。使用广泛宿主范围的接合辅助质粒，将oriT位点之间的区域接合到大肠杆菌受体菌株中，在其中将其环化并维持为嵌合mini-F载体。克隆的目标区域以多种方式功能化以适应下游操作。目标区域两侧是用于重组到其他载体中的 Gateway attB 位点，以及用于亚克隆的罕见 18 bp I-SceI 限制性位点。 Tn7 功能化靶标也可以插入天然存在的染色体 attTn7 位点，或作为广泛宿主范围的质粒维持，用于互补或异源表达研究。我们使用 oriTn7 捕获技术克隆和补充了伪鼻疽伯克霍尔德杆菌基因组区域，大小高达 140 kb，并创建了具有各种基因组岛组合的同基因伯克霍尔德杆菌菌株。我们相信该系统将极大地帮助基因组岛、生物合成基因簇和大型开放阅读框的克隆和遗传分析。
We have made significant improvements to a broad-host-range system for the cloning and manipulation of large bacterial genomic regions based on site-specific recombination between directly repeated oriT sites during conjugation. Using two suicide capture vectors carrying flanking homology regions, oriT sites are recombined on either side of the target region. Using a broad-host-range conjugation helper plasmid, the region between the oriT sites is conjugated into an Escherichia coli recipient strain, where it is circularized and maintained as a chimeric mini-F vector. The cloned target region is functionalized in multiple ways to accommodate downstream manipulation. The target region is flanked with Gateway attB sites for recombination into other vectors and by rare 18-bp I-SceI restriction sites for subcloning. The Tn7-functionalized target can also be inserted at a naturally occurring chromosomal attTn7 site(s) or maintained as a broad-host-range plasmid for complementation or heterologous expression studies. We have used the oriTn7 capture technique to clone and complement Burkholderia pseudomallei genomic regions up to 140 kb in size and have created isogenic Burkholderia strains with various combinations of genomic islands. We believe this system will greatly aid the cloning and genetic analysis of genomic islands, biosynthetic gene clusters, and large open reading frames.