We developed a method that allows rapid isolation of mutant Bacillus subtilis 168 carrying an inversion of a specific DNA segment of the genome. Two incomplete neomycin resistance gene cassettes were integrated at both ends of the 1652-kb segment to be inverted. Reciprocal recombination within the 590-bp homologous region of these two cassettes created an intact neomycin resistance gene with concomitant inversion of the 1652-kb segment flanked by the two cassettes. Structure of the mutant genome was verified by analyzing the physical map for rare cutting endonucleases, SfiI NotI, I-Ceul, and I-Scel. The inversion rate was estimated to be 6.9 ± 1.4 x 10 –8/cell/cell division at 37°C. The method should be in principle applicable not only to other regions of the B. subtilis genome but also to other bacterial genomes.
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