Figure 1

Construction of the recA -inducible BGM vector system. (a) The BEST310 and iREX constructs that possess two antibiotic resistance gene cassettes for BAC cloning: Pr-neo, a lambda Pr promoter fused to the neomycin resistance gene (neo), and cI-spc, which contains cI encoding the CI repressor protein, which binds to the Pr promoter, fused to the spectinomycin resistance gene (spc). The closed and open arrows indicate the BAC cloning site, and the red and blue lines indicate the pBR322 sequence. (b) The inducible recA expression cassette, pX-recA, was inserted at the amyE locus of the BEST310 genome via homologous recombination. amyE is not essential for the viability of B. subtilis [15]. cat, chloramphenicol acetyltransferase; H, HindIII; X, XhoI. (c) After introducing the pX-recA, the endogenous recA was replaced with the tetracycline resistance gene (tet) via homologous recombination. X, XhoI. (d) Southern blot analysis using an amyE probe indicated the correct insertion of pX-recA. The genomic DNA of the represented clones was digested with HindIII. The open arrowhead indicates the intact amyE in BEST310. The closed arrowheads indicate 5’-amyE and 3’-amyE divided by the insertion of pX-recA. (e) Southern blot analysis using a recA probe indicated the correct insertion of pCTP. The genomic DNA of the represented clones was digested with XhoI. The open arrowheads indicate the endogenous recA. The closed arrowheads indicate the inducible recA derived from pX-recA.