[Cloning and sequencing of promoter and signal sequence coding regions from Bacillus subtilis]

Promoter and signal sequence coding regions from B. subtilis were cloned in E. coli using a bifunctional and signal sequence selection plasmid pGPB14 as a vector. The Sau3A digested chromosome DNA was ligated with BamHI digested pGPB14. The ligated mixture was used to transform E. coli C600. Ampicillin and erythromycin resistant clones were selected. Recombinant plasmids were isolated from double resistant transformants. Restriction analysis showed that inserts of various length had been cloned and these inserts rendered the E. coli cells resistant to different concentrations of ampicillin. The recombinant plasmids were transformed and showed the same secretion function in B. subtilis. The amount and localization of beta-lactamase were determined in transformed E. coli and B. subtilis. The results indicate that the beta-lactamase activities of E. coli mainly in periplasm while the enzyme produced by B. subtilis secreted extracellularly. 10 of the cloned fragments were sequenced by Sanger's dideoxy chain termination method. The sequencing data show that all fragments contain promoter, ribosome binding site and signal sequence coding region.