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An Improved Method for oriT-Directed Cloning and Functionalization of Large Bacterial Genomic Regions

机译：大细菌基因组区域的定向克隆和功能化的改进方法

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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 Tn 7 -functionalized target can also be inserted at a naturally occurring chromosomal att Tn 7 site(s) or maintained as a broad-host-range plasmid for complementation or heterologous expression studies. We have used the oriTn 7 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.
机译：基于结合过程中直接重复的oriT位点之间的位点特异性重组，我们对克隆和操纵大型细菌基因组区域的广泛宿主系统进行了重大改进。使用携带侧翼同源性区域的两个自杀捕获载体，将oriT位点重组在靶区域的任一侧。使用宽宿主共轭辅助质粒，将oriT位点之间的区域缀合到大肠杆菌受体菌株中，并使其环化并保持为嵌合mini-F载体。克隆的靶区域以多种方式功能化以适应下游操作。靶区域的侧翼是Gateway attB位点以重组成其他载体，而罕见的18 bp I-SceI限制性位点则用于亚克隆。 Tn 7-功能化的靶标也可以插入天然存在的attn Tn 7位点或保持为广泛宿主范围的质粒用于互补或异源表达研究。我们已经使用oriTn 7捕获技术克隆和补充了大小不超过140 kb的假伯克霍尔德氏菌基因组区域，并创建了具有各种基因组岛组合的同基因伯克霍尔德菌菌株。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。

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