Rapid site site-directed domain scanning mutagenesis of enteropathogenic Escherichia coli espD

We developed a rapid mutagenesis method based on a modification of the QuikChange (R) system (Stratagene) to systemically replace endogenous gene sequences with a unique similar size sequence tag. The modifications are as follows: 1: the length of the anchoring homologous sequences of both mutagenesis primers were increased to 16 - 22 bp to achieve melting temperatures greater than 80 degrees C. 2: the final concentrations of both primers were increased to 5- 10 ng/mu l and the final concentration of template to 1-2 ng/mu l. 3: the annealing temperature was adjusted when necessary from 52 degrees C to 58 degrees C. We generated 25 sequential mutants in the cloned espD gene (1.2 kb), which encodes an essential component of the type III secretion translocon required for the pathogenesis of enteropathogenic E. coli (EPEC) infection. Each mutation consisted of the replacement of 15 codons (45 bp) with 8 codons representing a 24 bp sequence containing three unique restriction endonuclease sites (KpnI/MfeI/SpeI) starting from the second codon. The insertion of the restriction endonuclease sites provides a convenient method for further insertions of purification and/or epitope tags into permissive domains. This method is rapid, site-directed and allows for the systematic creation of mutants evenly distributed throughout the entire gene of interest.