School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK.
BMC Microbiol. 2009 Dec 9;9:252. doi: 10.1186/1471-2180-9-252.
Homologous recombination mediated by the lambda-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 lambda-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 lambda-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains.
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 lambda-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 6xHis, 3xFLAG, 4xProteinA 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 lambda-Red system, which can lead to unwanted secondary alterations to the chromosome.
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.
由 lambda-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。已经开发了几种在将携带与染色体同源的区域的 DNA 递送入细胞的机制上有所不同的方案。一种常见的技术是将线性 DNA 片段电穿孔到细胞中。或者，通过用不会切割宿主基因组的核酸内切酶消化供体质粒，在体内生成 DNA 片段。在这两种情况下，lambda-Red 基因产物都将线性 DNA 片段上携带的同源区域与染色体进行重组。我们已经成功地将这两种技术用于在大肠杆菌 K-12 菌株中生成染色体突变。然而，我们在致病性大肠杆菌菌株中使用这些基于 lambda-Red 的重组技术的效果有限，这促使我们为这些菌株中的重组工程开发了一种增强的方案。
我们的目标是开发一种高通量的重组工程系统，主要用于将基因与表位标签偶联，也可用于删除致病性和 K-12 大肠杆菌菌株中的基因。为此，我们设计了一系列用于 lambda-Red 重组系统的供体质粒，当在体内用 I-SceI 核酸内切酶切割时，会产生离散的线性 DNA 片段，允许在染色体基因的 C 末端添加 6xHis、3xFLAG、4xProteinA 或 GFP 标签，或删除染色体区域。我们通过包含赋予卡那霉素抗性的盒以及在供体质粒上包含 sacB 基因来增强现有的方案和技术，从而使得除真正的重组子之外的所有物质都在含有卡那霉素和蔗糖的培养基中被反向选择，从而消除了对广泛筛选的需求。这种方法还有一个额外的优势，即限制了细胞暴露于 lambda-Red 系统潜在的有害影响，这可能导致染色体的不必要的二次改变。
我们已经开发了一种针对大肠杆菌的表位标记或基因删除的反向选择重组工程技术。我们通过修饰肠出血性 O157:H7（EHEC）、尿路致病性 CFT073（UPEC）、肠聚集性 O42（EAEC）和肠毒性 H10407（ETEC）大肠杆菌菌株以及 K-12 实验室菌株的染色体，证明了该技术的多功能性。
