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首页> 外文期刊>BMC Microbiology >Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains
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Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

机译：基因调控：在实验室和致病性大肠杆菌菌株中重组的方法

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Background Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains. Results Our goal was to develop a high-throughput recombineering system, primarily for the coupling of genes to epitope tags, which could also be used for deletion of genes in both pathogenic and K-12 E. coli strains. To that end we have designed a series of donor plasmids for use with the λ-Red recombination system, which when cleaved in vivo by the I-SceI meganuclease generate a discrete linear DNA fragment, allowing for C-terminal tagging of chromosomal genes with a 6 × His, 3 × FLAG, 4 × ProteinA or GFP tag or for the deletion of chromosomal regions. We have enhanced existing protocols and technologies by inclusion of a cassette conferring kanamycin resistance and, crucially, by including the sacB gene on the donor plasmid, so that all but true recombinants are counter-selected on kanamycin and sucrose containing media, thus eliminating the need for extensive screening. This method has the added advantage of limiting the exposure of cells to the potential damaging effects of the λ-Red system, which can lead to unwanted secondary alterations to the chromosome. Conclusion We have developed a counter-selective recombineering technique for epitope tagging or for deleting genes in E. coli. We have demonstrated the versatility of the technique by modifying the chromosome of the enterohaemorrhagic O157:H7 (EHEC), uropathogenic CFT073 (UPEC), enteroaggregative O42 (EAEC) and enterotoxigenic H10407 (ETEC) E. coli strains as well as in K-12 laboratory strains.
机译：背景技术由λ-Red基因介导的同源重组是在大肠杆菌中进行染色体修饰的常用方法。已经开发出几种方案，其机制不同，将携带与染色体同源的区域的DNA传递到细胞中的机制不同。一种常见的技术是将线性DNA片段电穿孔到细胞中。或者，通过用不切割宿主基因组的核酸酶消化供体质粒，在体内产生DNA片段。在这两种情况下，λ-Red基因产物重组带有染色体的线性DNA片段上携带的同源区域。我们已经成功地使用了两种技术来在大肠杆菌K-12菌株中产生染色体突变。但是，我们在致病性大肠杆菌菌株中使用基于λ-Red的重组技术取得的成功有限，这导致我们开发了用于此类菌株重组的增强方案。结果我们的目标是开发一种高通量重组系统，主要用于基因与表位标签的偶联，该系统还可用于致病性和K-12大肠杆菌菌株中的基因缺失。为此，我们设计了一系列供λ-Red重组系统使用的供体质粒，当被I-SceI大范围核酸酶在体内切割时，会产生离散的线性DNA片段，从而可以用C-末端标记染色体基因。 6×His，3×FLAG，4×ProteinA或GFP标签，或用于删除染色体区域。我们已经通过包含赋予卡那霉素抗性的盒，并且最重要的是，在供体质粒上包括sacB基因，增强了现有的协议和技术，从而在卡那霉素和含蔗糖的培养基上反选择了除真重组子以外的所有重组子，从而消除了需要进行广泛的筛选。该方法的另一个优点是可以限制细胞对λ-Red系统的潜在破坏作用的暴露，这可能会导致染色体发生不必要的二次改变。结论我们开发了一种针对表位标签或删除大肠杆菌中基因的反选择性重组技术。我们已经通过修改肠出血性O157：H7（EHEC），尿路致病性CFT073（UPEC），肠聚集性O42（EAEC）和肠毒素H10407（ETEC）大肠杆菌菌株以及K-12的染色体证明了该技术的多功能性实验室菌株。

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