Generation of Isogenic Deletion Mutants of STEC

Genetic tools are necessary to unravel complex phenotypes, like the formation of attaching and effacing lesions by Shiga toxin-producing Escherichia coli (STEC). To inactivate a particular gene, we use a precise and convenient method that is based on the generation of an in-frame deletion within this gene. The advantages of this technique in comparison with others (e.g., transposon mutagenesis or site-directed insertional mutagenesis) are as follows:

–	No polar effects on the transcription of downstream located genes by disrupting polycistronic RNA transcripts.
–	Genetic stability of the generated deletion mutants, because a reversion to the wild-type is impossible.
–	No continuous selective pressure is needed to maintain the mutant.