title: Integrative bacterial artificial chromosomes for DNA integration into the Bacillus subtilis chromosome.
abstract: Bacillus subtilis is a well-characterized model bacterium frequently used for a number of biotechnology and synthetic biology applications. Novel strategies combining the advantages of B. subtilis with the DNA assembly and editing tools of Escherichia coli are crucial for B. subtilis engineering efforts. We combined Gibson Assembly and λ red recombineering in E. coli with RecA-mediated homologous recombination in B. subtilis for bacterial artificial chromosome-mediated DNA integration into the well-characterized amyE target locus of the B. subtilis chromosome. The engineered integrative bacterial artificial chromosome iBAC(cav) can accept any DNA fragment for integration into B. subtilis chromosome and allows rapid selection of transformants by B. subtilis-specific antibiotic resistance and the yellow fluorescent protein (mVenus) expression. We used the developed iBAC(cav)-mediated system to integrate 10kb DNA fragment from E. coli K12 MG1655 into B. subtilis chromosome. iBAC(cav)-mediated chromosomal integration approach will facilitate rational design of synthetic biology applications in B. subtilis.
authors.affiliation: Department of Pathology, University of Cambridge, Tennis Court Road, CB2 1QP Cambridge, UK. Electronic address: mj417@cam.ac.uk.
authors.forename: Mario
authors.initials: M
authors.lastname: Juhas
authors.affiliation: Department of Pathology, University of Cambridge, Tennis Court Road, CB2 1QP Cambridge, UK.
authors.forename: James W
authors.initials: JW
authors.lastname: Ajioka
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics*; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D002876:Chromosomes, Bacterial; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D010957:Plasmids; D011938:Rec A Recombinases / Q000235:genetics*; D011995:Recombination, Genetic; D058615:Synthetic Biology / Q000379:methods; D014169:Transformation, Bacterial
chemical_list: D011938:Rec A Recombinases
keywords: Bacterial artificial chromosome; Chromosomal integration; RecA homologous recombination; mVenus; λ red recombineering