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.
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Acknowledgments
The authors acknowledge the financial support of the UK Medical Research Council (G0601176) and the UK Biotechnology and Biological Sciences Research Council (BB/L013940/1 and BB/G016224/1).
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Ehsaan, M., Kuehne, S.A., Minton, N.P. (2016). Clostridium difficile Genome Editing Using pyrE Alleles. In: Roberts, A., Mullany, P. (eds) Clostridium difficile. Methods in Molecular Biology, vol 1476. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6361-4_4
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DOI: https://doi.org/10.1007/978-1-4939-6361-4_4
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