Figure 3

Verification of the correct integration of the two gene clusters and bacillomycin production in B. subtilis 1A751 + sfp.

(A) Schematic presentation of gene cluster integration in B. subtilis 1A751 + sfp and PCR setup. (B). Detection correct clones of insertion of ede gene cluster in B. subtilis 1A751 + sfp by 5′ and 3′ junction PCR. This figure present the method of screening the correct transformants. The fragment of 5′ junction PCR indicates that the gene cluster was correctly integrated in the amyE-F locus. And the fragment of 3′ junction PCR indicates that the gene cluster was correctly integrated in the amyE-R locus. The correct transformants should have both 5′ junction PCR fragment and 3′ junction PCR fragment. Lane M is the Takara 1kb ladder. Lane N is the host stain as negative control. Lanes 1–10 are recombinants. The predicted size of the PCR products of 5′ and 3′ junction PCR are 1958 bp and 2769 bp, respectively. All ten clones are correct transformant resulting from double crossover. (C). Detection of the correct clones of bmy gene cluster in B. subtilis 1A751 + sfp by 5′ and 3′ junction PCR. The predicted size of the PCR products of 5′ and 3′ junction PCR are 2147 bp and 3047 bp, respectively. Clone 2 and clone 10 are correct transformant resulting from double crossover. Clones 3/4/5/6/8 might result from single crossover. (D). HPLC-MS analysis of bacillomycin production in B. subtilis 1A751 + sfp. (E). MS spectra of compounds 1, 2 and 3.Compound 1 indicates C14-bacillomycin D (m/z 1031.5391 [M + H]⁺and 1053.5208 [M + Na]⁺). Compound 2 indicates C15-bacillomycin D (m/z 1045.5546 [M + H]⁺and 1067.5366 [M + Na]⁺). Compound 3 indicates C16-bacillomycin D (m/z 1059.5706 [M + H]⁺).