title: Clostridium difficile Genome Editing Using pyrE Alleles.
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.
authors: Clostridia Research Group, BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK.
authors: Muhammad
authors: M
authors: Ehsaan
authors: Clostridia Research Group, BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK.|Nottingham Digestive Disease Centre, NIHR Biomedical Research Unit, The University of Nottingham, University Park, Nottingham, UK.
authors: Sarah A
authors: SA
authors: Kuehne
authors: Clostridia Research Group, BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK. nigel.minton@nottingham.ac.uk.|Nottingham Digestive Disease Centre, NIHR Biomedical Research Unit, The University of Nottingham, University Park, Nottingham, UK. nigel.minton@nottingham.ac.uk.
authors: Nigel P
authors: NP
authors: Minton
mesh_terms: D000483:Alleles; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D016360:Clostridioides difficile / Q000235:genetics*; D043525:Cytosine Deaminase / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D000072669:Gene Editing / Q000379:methods*; D015870:Gene Expression; D005816:Genetic Complementation Test; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D016680:Genome, Bacterial; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009962:Orotate Phosphoribosyltransferase / Q000235:genetics*; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D009962:Orotate Phosphoribosyltransferase; D043525:Cytosine Deaminase
keywords: Allele-coupled exchange (ACE); Allelic exchange; Clostridium difficile; Complementation; Counterselection marker; Overexpression; Pseudo-suicide; codA; pyrE