[Abstract]  [Full Text]  [PDF]

Qiwen Deng², Wensheng Luo³ and Michael S. Donnenberg^1*

¹ Division of Infectious Diseases, Department of Medicine, University of Maryland School of Medicine. 20 Penn Street, Baltimore, Maryland, 21201. USA.
² Division of Infectious Diseases, Department of Medicine, University of Maryland School of Medicine, 20 Penn Street, Baltimore, Maryland, 21201, USA. Current address: Division of Infectious Disease, the Affiliated Shenzhen Nanshan Hospital of Guangdong Medical College. Deng and Luo contributed to this work equally and are listed in alphabetical order. Shenzhen 518052. P.R. China.
³ Division of Infectious Diseases, Department of Medicine, University of Maryland School of Medicine. Deng and Luo contributed to this work equally and are listed in alphabetical order. 20 Penn Street, Baltimore, Maryland, 21201. USA.

* To whom correspondence should be addressed: Michael S. Donnenberg, Division of Infectious Diseases, Department of Medicine, University of Maryland School of Medicine. 20 Penn Street, Baltimore, Maryland, 21201. USA. Email: mdonnenb@umaryland.edu

Biol. Proced. Online 2007;9:18-26. doi:10.1251/bpo130
Submitted: March 20, 2007; Accepted: July 13, 2007; Published: September 14, 2007.

Indexing terms: Mutagenesis, Site-Directed; Polymerase Chain Reaction; Plasmids; Sequence Deletion.

Abbreviations: bp, base pair; EPEC, enteropathogenic Escherichia coli; %G+C, percentage guanine plus cytosine DNA content; kb, kilobase; PAGE, polyacrylamide gel electrophoresis; Tm, melting temperature.

We developed a rapid mutagenesis method based on a modification of the QuikChange® 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°C. 2: the final concentrations of both primers were increased to 5-10 ng/μl and the final concentration of template to 1-2 ng/μl. 3: the annealing temperature was adjusted when necessary from 52°C to 58°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.