Fig. 3

The construction of a Bacillus integrative plasmid (BIP) and a BIP combining a synthetic gene circuit (BIPS) to delete the aprE gene of B. pumilus. A The pSGC2iN-Pmapr for B. pumilus aprE gene deletion via the BIP system was constructed by cloning homologous arms (Pm-N and Pm-C) and chloramphenicol resistance gene (cat) to pSGC2iN. B The construction of pSGC4iN-Pmapr to delete the aprE gene of B. pumilus via the BIPS system. The synthetic gene circuit of pSGC4iN-Pmapr consists of xylR-P_xylA-lacI and PmN-P_spac-cat-PmC cassettes that enhance recipient mutation selection. C The scheme of the synthetic gene circuit to function as a counter-selectable marker. In the absence of xylose, the XylR repressor blocks lacI gene expression and the cat gene is expressed, rendering the recipient resistant to chloramphenicol. In contrast, the addition of xylose induces lacI expression, which suppresses cat gene expression, making cells sensitive to chloramphenicol. D Structure of target chromosome when BIPS is inserted through single crossover integration (SCO). Transformants with the entire plasmid integrated into the chromosome by SCO can survive in the absence of xylose and the presence of chloramphenicol. E Induction of in vivo recombination from SCO colonies. The survival of cells containing the entire plasmid in the presence of xylose and chloramphenicol requires the deletion of the P_xyl-lacI cassette by in vivo recombination between homologous C-terminal fragments. In vivo recombination will generate genomic mutations