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James R. Emmerson¹, David L. Gally¹ and Andrew J. Roe^1*

¹ Centre for Infectious Diseases, University of Edinburgh. The Chancellors Building, 49 Little France Crescent, EH16 4SB. United Kingdom.

* To whom correspondence should be addressed: Andrew J. Roe, Centre for Infectious Diseases, University of Edinburgh. The Chancellors Building, 49 Little France Crescent, EH16 4SB. United Kingdom. Email: aroe@vet.ed.ac.uk

Biol. Proced. Online 2006;8:153-162. doi:10.1251/bpo123
Submitted: July 05, 2006; Accepted: September 12, 2006; Published: September 27, 2006.

Indexing terms: Gene Deletion; Escherichia coli O157:H7.

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.