Clostridium difficile Genome Editing Using pyrE Alleles

Muhammad Ehsaan, Sarah Kuehne, Nigel P Minton

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5 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)35-52
Number of pages18
JournalMethods in molecular biology
Early online date10 Aug 2016
Publication statusE-pub ahead of print - 10 Aug 2016


  • Journal Article


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