Generation of gene deletions and gene replacements in Escherichia coli O157:H7 using a temperature sensitive allelic exchange system

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.