Pulse-field gel-electrophoretic analysis of the amplification and copy-number stability of an integrational plasmid in Bacillus subtilis

The stability of integration and amplification of an integrational plasmid in Bacillus subtilis was analyzed. A cat-containing plasmid was constructed that could be integrated into the amy locus to facilitate measurement of excision events. Pulse-field gel electrophoresis was used to measure the copy number in strains that were resistant to different levels of chloramphenicol. The stability of the amplified unit in strains containing from 2 to 18 tandem copies of the amplicon in the presence and absence of chloramphenicol and through different generation times was then determined. Our results demonstrate that, for any given strain, the copy number of the amplicon remains stable. Furthermore, this stability is maintained when a clone containing an amplicon of defined size is cultured through as many as 100 generations in the absence of selective pressure.