Cloning of DNA fragments with promoter function from temperate phage of Bacillus licheniformis

Phage Blp7 DNA was digested with restriction enzyme and ligated to the restriction enzyme digested vector pTG402. The ligated mixture was used to transform competent cells of E. coli MC1061. Plasmid DNA was extracted from pooled transformants and competent cells of B. subtilis were transformed. By selecting yellow colonies upon spraying with catechol solution, 22 clones containing DNA fragments with promoter function were obtained. The promoter activity of 15 clones was determined by the color reaction of catechol-2,3-dioxygenase. The inserted fragment of the most potent promoter was mapped with restriction enzymes. CatO2 ase activity of two clones was measured in cells of B. subtilis of all growth phases and was found to increase rapidly at the end of the log-phase. It is inferred that these two promoters might be recognized by sigma 37.