Clostridium difficile Genome Editing Using pyrE Alleles

  • Muhammad Ehsaan
  • Sarah A. Kuehne
  • Nigel P. Minton
Part of the Methods in Molecular Biology book series (MIMB, volume 1476)


Precise manipulation (in-frame deletions and substitutions) of the Clostridium difficile genome is possible through a two-stage process of single-crossover integration and subsequent isolation of double-crossover excision events using replication-defective plasmids that carry a counterselection marker. Use of a codA (cytosine deaminase) or pyrE (orotate phosphoribosyltransferase) as counter selection markers appears equally effective, but there is considerable merit in using a pyrE mutant as the host as, through the use of allele-coupled exchange (ACE) vectors, mutants created (by whatever means) can be rapidly complemented concomitant with restoration of the pyrE allele. This avoids the phenotypic effects frequently observed with high-copy-number plasmids and dispenses with the need to add antibiotic to ensure plasmid retention.

Key words

Clostridium difficile Pseudo-suicide Allelic exchange Allele-coupled exchange (ACE) Counterselection marker pyrE codA Complementation Overexpression 


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Muhammad Ehsaan
    • 1
  • Sarah A. Kuehne
    • 1
    • 2
  • Nigel P. Minton
    • 1
    • 2
  1. 1.Clostridia Research Group, BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), School of Life Sciences, Centre for Biomolecular SciencesUniversity of NottinghamNottinghamUK
  2. 2.Nottingham Digestive Disease Centre, NIHR Biomedical Research UnitThe University of NottinghamNottinghamUK

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