title: Development of an efficient technique for gene deletion and allelic exchange in Geobacillus spp.
abstract: BACKGROUND
  Geobacillus thermoglucosidasius is a thermophilic, natural ethanol producer and a potential candidate for commercial bioethanol production. Previously, G. thermoglucosidasius has been genetically modified to create an industrially-relevant ethanol production strain. However, creating chromosomal integrations and deletions in Geobacillus spp. is laborious. Here we describe a new technique to create marker-less mutations in Geobacillus utilising a novel homologous recombination process.
  
  
  RESULTS
  Our technique incorporates counter-selection using β-glucosidase and the synthetic substrate X-Glu, in combination with a two-step homologous recombination process where the first step is a selectable double-crossover event that deletes the target gene. We demonstrate how we have utilised this technique to delete two components of the proteinaceous shell of the Geobacillus propanediol-utilization microcompartment, and simultaneously introduce an oxygen-sensitive promoter in front of the remaining shell-component genes and confirm its functional incorporation.
  
  
  CONCLUSIONS
  The selectable deletion of the target gene in the first step of our process prevents re-creation of wild-type which can occur in most homologous recombination techniques, circumventing the need for PCR screening to identify mutants. Our new technique therefore offers a faster, more efficient method of creating mutants in Geobacillus.
authors.affiliation: Department of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, UK.
authors.forename: Leann F
authors.initials: LF
authors.lastname: Bacon
authors.affiliation: Department of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, UK.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Hamley-Bennett
authors.affiliation: Department of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, UK.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Danson
authors.affiliation: Department of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, UK. d.j.leak@bath.ac.uk.
authors.forename: David J
authors.initials: DJ
authors.lastname: Leak
mesh_terms: D000483:Alleles; D000431:Ethanol / Q000378:metabolism; D017353:Gene Deletion; D005818:Genetic Engineering / Q000379:methods*; D056506:Geobacillus / Q000235:genetics*; D059765:Homologous Recombination; D009154:Mutation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D017384:Sequence Deletion; D001617:beta-Glucosidase / Q000378:metabolism
chemical_list: D000431:Ethanol; D001617:beta-Glucosidase