Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains.
Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains.
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DOI：
10.1186/1471-2180-9-252
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发表时间：
2009-12-09
期刊：
影响因子：
4.2
通讯作者：
Hobman JL
中科院分区：
文献类型：
--
作者：
Lee DJ;Bingle LE;Heurlier K;Pallen MJ;Penn CW;Busby SJ;Hobman JL
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。已经开发出多种方案，其机制不同，携带与染色体同源区域的 DNA 被递送到细胞中。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 基因产物将线性 DNA 片段上携带的同源区域与染色体重组。我们已成功使用这两种技术在大肠杆菌 K-12 菌株中产生染色体突变。然而，我们在致病性大肠杆菌菌株中使用这些基于 λ-Red 的重组技术取得的成功有限，这促使我们开发了一种用于此类菌株重组工程的增强方案。我们的目标是开发一种高通量重组工程系统，主要用于将基因与表位标签偶联，该系统也可用于删除致病性和 K-12 大肠杆菌菌株中的基因。为此，我们设计了一系列与 λ-Red 重组系统一起使用的供体质粒，当这些质粒在体内被 I-SceI 大范围核酸酶切割时，会生成离散的线性 DNA 片段，从而可以使用 6 × His、3 × FLAG、4 × ProteinA 或 GFP 标签对染色体基因进行 C 末端标记，或用于删除染色体区域。我们通过包含赋予卡那霉素抗性的盒，以及最重要的是，通过在供体质粒上包含 sacB 基因，增强了现有的方案和技术，以便在含有卡那霉素和蔗糖的培养基上对除真正重组体之外的所有重组体进行反选择，从而消除了广泛筛选的需要。该方法的另一个优点是限制细胞暴露于 λ-Red 系统的潜在破坏性影响，这可能导致染色体发生不必要的二次改变。我们开发了一种反选择性重组技术，用于在大肠杆菌中进行表位标记或删除基因。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) 和肠产毒性 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。
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. 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. 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.
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影响因子：
14.9
作者：
Butala M;Busby SJ;Lee DJ
通讯作者：
Lee DJ
影响因子：
--
作者：
Murphy KC;Campellone KG
通讯作者：
Campellone KG
DOI：
10.1073/pnas.121164898
发表时间：
2001-06-05
影响因子：
11.1
作者：
Ellis, HM;Yu, DG;Court, DL
通讯作者：
Court, DL
影响因子：
3.5
作者：
SCHWEIZER, HP;HOANG, TT
通讯作者：
HOANG, TT
影响因子：
--
作者：
Poteete AR;Fenton AC;Nadkarni A
通讯作者：
Nadkarni A