Generation of gene deletions and gene replacements in Escherichia coli O157:H7 using a temperature sensitive allelic exchange system
Generation of gene deletions and gene replacements in Escherichia coli O157:H7 using a temperature sensitive allelic exchange system
复制标题
DOI：
10.1251/bpo123
复制
发表时间：
2006-09-27
影响因子：
6.4
通讯作者：
Roe, Andrew J.
中科院分区：
文献类型：
--
作者：
Emmerson, James R.;Gally, David L.;Roe, Andrew J.
在这项工作中，我们描述了在大肠杆菌O157:H7中使用温度敏感质粒产生基因缺失和基因替换的方案。这项技术需要将侧翼DNA克隆到对温度敏感的载体中，但由此产生的克隆为进一步修改目标序列提供了很大的灵活性。因此，它非常适合于设想发生几轮变化的基因研究。许多例子被用来说明该系统的灵活性，该系统已被用于创建新的基因替代，包括用于蛋白质定位工作的融合和用于转录分析的报告。在本文中，我们描述了可用于大肠杆菌O157时具有高度成功的协议。详细介绍了大肠杆菌O157 LEE4操纵子的缺失和替换，以显示该技术的优势和局限性。
In this work we describe protocols for the generation of gene deletions and gene replacements using a temperature sensitive plasmid in Escherichia coli O157:H7. This technology requires flanking DNA to be cloned into a temperature sensitive vector but the resulting clone allows great flexibility for further modification of the target sequence. It is therefore highly suited to the study of genes in which several rounds of changes are envisaged. A number of examples are used to illustrate the flexibility of the system which has been used to create novel gene replacements including fusions for protein localisation work and reporters for transcriptional analyses. In this paper we describe protocols which can be used with a high degree of success when applied to E. coli O157. The deletion and replacement of the LEE4 operon of E. coli O157 is detailed to show the advantages and limitations of the technology.