FIG. 2.

Vectors containing antibiotic resistance marker cassettes (A) and scheme for Cre/lox and PCR-based mutation delivery in B. subtilis (B). (A) ori, ColE1 replication origin; amp, Amp^r marker; zeo/spc, Zeo^r or Spc^r marker; horizontal solid arrows, primer binding site. (B) (i) lox71-spc/zeo-lox66,lox71-spc-lox66/lox71-zeo-lox66 cassette. The front and back regions flanking the target to be deleted are PCR amplified, gel purified and fused by PCR. (ii) PCR-fused products are directly used to transform B. subtilis, and Spc^r or Zeo^r transformants are selected. (iii) pTSC is introduced into a Spc^r or Zeo^r clone, and the constitutively expressed Cre recombinase mediates the recombination between lox71 and lox66. (iv) pTSC is eliminated to get the target strain by patching a transformant on antibiotic-free LB agar and incubating it at 51°C.