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.
Highlights
The ability to generate directed gene deletions and gene replacements is a central technique used to demonstrate gene function and fulfil Molecular Koch’s postulates in any study [1]
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
In this paper we describe protocols which can be used with a high degree of success when applied to E. coli O157
Summary
The ability to generate directed gene deletions and gene replacements is a central technique used to demonstrate gene function and fulfil Molecular Koch’s postulates in any study [1]. The phage λ red recombinase has been successfully applied to allow rapid, one step inactivation of genes [4]. These techniques can suffer from the requirement of specific genetic backgrounds (often mutants themselves), and are optimized for efficient use in E. coli K-12 laboratory strains. The application of these techniques to “wildtype” strains including pathotypes of E. coli is, often problematic. Lambda red is probably the most rapid system for inactivation of genes it does suffer from the fact that transient expression of the recombinase system enhances the frequency of unwanted genetic rearrangements. As the temperature sensitive allelic exchange system does not rely on any conjugation step, for example when using lambda Pirdependent iroR6K plasmids, strains that produce bacteriocins or capsule can be successfully modified
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