Figure 3. Scheme for mutation delivery procedure in B. subtilis.

A. (a) For purpose of gene point-mutation, the upstream and downstream fragments carrying the mutation site (*) were amplified by PCR with primer pairs P1/P2 and P3/P4, and upp-cassette was amplified by PCR with primer pairs P5/P6, respectively. (b) A triple fusion PCR reaction joined them with the upp-cassette. (c) PCR-fused product was used directly to transform BUK, and integration of the upp-cassette by first double-crossover event yields 5FU^S Cm^R transformants. (d) The shuttle vector pEBS-cop1 was transformed into the 5FU^S Cm^R strain to cleavage of the chromosome at the recognition site by I-SceI under xylose induction. (e) The excision of upp-cassette through the single-crossover event between the 30 bp DR stimulated by DSB generated a 5FU^R Cm^S strain that carried only the desired mutation without any other modification in the chromosome. B. For gene deletion, the upstream and downstream fragments carrying the same DR sequence (*) were amplified by PCR with primer pairs P7/P9 and P10/P8. The remainder of the procedure was essentially the same as above described.