Generation of gene deletions and gene replacements in Escherichia coli O157:H7 using a temperature sensitive allelic exchange system
- 1 December 2006
- journal article
- Published by Springer Science and Business Media LLC in Biological Procedures Online
- Vol. 8 (1), 153-162
- https://doi.org/10.1251/bpo123
Abstract
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.Keywords
This publication has 15 references indexed in Scilit:
- Analysis of fimbrial gene clusters and their expression in enterohaemorrhagic Escherichia coli O157:H7Environmental Microbiology, 2006
- A comparison of enteropathogenic and enterohaemorrhagicEscherichia colipathogenesisFEMS Microbiology Letters, 2006
- Co‐ordinate single‐cell expression of LEE4‐ and LEE5‐encoded proteins of Escherichia coli O157:H7Molecular Microbiology, 2004
- EspFU Is a Translocated EHEC Effector that Interacts with Tir and N-WASP and Promotes Nck-Independent Actin AssemblyDevelopmental Cell, 2004
- Molecular Koch's postulates applied to bacterial pathogenicity — a personal recollection 15 years laterNature Reviews Microbiology, 2004
- Heterogeneous Surface Expression of EspA Translocon Filaments by Escherichia coli O157:H7 Is Controlled at the Posttranscriptional LevelInfection and Immunity, 2003
- Tools for Characterization of Escherichia coli Genes of Unknown FunctionJournal of Bacteriology, 2002
- Genome sequence of enterohaemorrhagic Escherichia coli O157:H7Nature, 2001
- Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature‐sensitive pSC101 repliconMolecular Microbiology, 1991
- A simple and rapid method to obtain substitution mutations in Escherichia coli: isolation of a dam deletion/insertion mutationGene, 1988