Bacillus subtilis 로부터 ampicillin 저항성을 지니는 plasmid 의 분리 및 특성 분석

Bacillus subtilis strains were screened for the resistance to antibiotics. Some of the strains were resistant to gramicidin S and streptomycin. Particularly, the wild (trp+) strain was resistant to ampicillin and penicillin-G. Plasmids in these strains were detected and isolated by the methods of both Birnboim (1979) and Kado (1981). Plasmids were detected only in the wild (trp+) strain. The plasmids were designated as pBL100 and estimated to be 50Kb to 60Kb. Sodium dodecyl sulfate (SDS) was used for curing. The wild (trp+) strain did not grow when SDS concentration was higher than 0.00275% (w/v). The bacterial cells grown at this concentration were screened for the resistance to ampicillin. Two colonies were found to be sensitive to ampicillin. Plasmid was not detected in the cells from these colonies. Minimum concentration of antibiotics inhibitory to the growth of the cured cells was determined. The cured cells showed reduced resistance to ampicillin. The transformation of plasmids was made according to the protoplast method. When ATCC 6633 and ATCC 23857 were used as recipients, a colony for each strain was produced on the medium containing 50㎍/ml ampicillin. The plasmids in the cells of these colonies were found to be the same as those in the wild (trp+) strain. When 168 (trp-) strain was used as a recipient, 141 out of 180 colonies screened did grow on the medium containing 100μg/ml ampicillin. The plasmids in the cells of these colonies were also found to be the same as those in the wild (trp+) strain. Minimum inhibitory concentration of antibiotics for the transformants was similar to that of the wild (trp+) strain