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Biological Procedures Online

, Volume 9, Issue 1, pp 18–26 | Cite as

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

Open Access
Article

Abstract

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.

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

References

  1. 1.
    Biery MC, Stewart FJ, Stellwagen AE, Raleigh EA, Craig NL. A simple in vitro Tn7-based transposition system with low target site selectivity for genome and gene analysis. Nucleic Acids Res 2000; 28:1067–1077.PubMedCrossRefGoogle Scholar
  2. 2.
    Anton BP, Raleigh EA. Transposon-mediated linker insertion scanning mutagenesis of the Escherichia coli McrA endonuclease. J Bacteriol 2004; 186:5699–5707.PubMedCrossRefGoogle Scholar
  3. 3.
    Shevchenko Y, Bouffard GG, Butterfield YS, Blakesley RW, Hartley JL, Young AC et al. Systematic sequencing of cDNA clones using the transposon Tn5. Nucleic Acids Res 2002; 30:2469–2477.PubMedCrossRefGoogle Scholar
  4. 4.
    Luo W, Donnenberg MS. Analysis of the function of enteropathogenic Escherichia coli EspB by random mutagenesis. Infect Immun 2006; 74:810–820.PubMedCrossRefGoogle Scholar
  5. 5.
    Donnenberg MS, Yu J, Kaper JB. A second chromosomal gene necessary for intimate attachment of enteropathogenic Escherichia coli to epithelial cells. J Bacteriol 1993; 175:4670–4680.PubMedGoogle Scholar
  6. 6.
    Lai LC, Wainwright LA, Stone KD, Donnenberg MS. A third secreted protein that is encoded by the enteropathogenic Escherichia coli pathogenicity island is required for transduction of signals and for attaching and effacing activities in host cells. Infect Immun 1997; 65:2211–2217.PubMedGoogle Scholar
  7. 7.
    Owen RJ, Hill LR, Lapage SP. Determination of DNA base compositions from melting profiles in dilute buffers. Biopolymers 1969; 7:503–516.PubMedCrossRefGoogle Scholar
  8. 8.
    Ellington A, Pollard JD. Jr. Purification of Oligonucleotides Using Denaturing Polyacrylamide Gel Electrophoresis. In Current Protocols in Molecular Biology. Edited by Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA et al. New York: John Wiley and Sons; 1998:2.12.1–2.12.7.Google Scholar

Copyright information

© Springer 2007

Authors and Affiliations

  • Qiwen Deng
    • 1
  • Wensheng Luo
    • 1
  • Michael S. Donnenberg
    • 1
  1. 1.Division of Infectious Diseases, Department of MedicineUniversity of Maryland School of MedicineBaltimoreUSA

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