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Clostridium difficile

Volume 1476 of the series Methods in Molecular Biology pp 35-52

Date:

Clostridium difficile Genome Editing Using pyrE Alleles

  • Muhammad EhsaanAffiliated withClostridia Research Group, BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham
  • , Sarah A. KuehneAffiliated withClostridia Research Group, BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), School of Life Sciences, Centre for Biomolecular Sciences, University of NottinghamNottingham Digestive Disease Centre, NIHR Biomedical Research Unit, The University of Nottingham
  • , Nigel P. MintonAffiliated withClostridia Research Group, BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), School of Life Sciences, Centre for Biomolecular Sciences, University of NottinghamNottingham Digestive Disease Centre, NIHR Biomedical Research Unit, The University of Nottingham Email author 

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Abstract

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