Cloning of DNA fragments which promte gene expression in bacillus subtilis system

Abstract

To understand the transcription initiation mechanism in psychrophilic bacilli and achieve a DNA fragment exerting strong promoter activity in B.stbtilis, a B.subtilis/E. coli promoter probe shuttle vector by ligation of promoterless cat-86 gene and B. subtilis/E. coli shuttle vector, pZ124 has been constructed. This plasmid, pZC101, was stably maintained in both species but only kanamycin resistance gene was expressed and sensitive to 30 $\mu$g/ml chloramphenicol in Bacillus subtilis. Using this promoter probe vector, 55 promoter clones from two Bacillus species, B. subtilis JU70 and B. insolitus ATCC23299(psychrophile) were obtained. By examination of chloramphenicol resistance on solid media, SDS-PAGE analysis, RNA dot-blots, and CAT specific activity, some of the promoter clone, such as pI1 have 3 fold higher CAT specific activity than control promoter SPOII at 37$^\circ$C growth.