한국 재래식 된장.간장 발효균 Bacillus sp. SSA3 균주의 expression vector를 개발하기 위해 Bacillus sp. SSA3의 chromosomal DNA로부터 유전자의 promoter 부위를 cloning 하였다. Recombinant plasmid를 제작하기 위하여 Bacillus sp. SSA3의 chromosomal DNA을 HindIII로 절단한 단편을 pGR71 plasmid의 CAT gene과 pUC18 plasmid의 $eta$-galactosidase gene의 전방에 삽입시킨 후, E.coli JM109에 형질전환하였다.E. coli JM109의 chloramphenicol 내성 clones으로부터 6 recombinant plasmid를 선별하였다. 이들 선별된 plasmid는 Bacillus sp. SSA3의 expression vector로 사용시 각 재조합 plasmid 중에 삽입된 promoter의 염에 대한 영향의 정도를확인하기 위해 10% NaCl이 첨가된 LB medium상에서 배양하였을 때, 이들 중 Bacillus sp. SSA3의 4 clones은 융합 CAT gene의 발현이 강하게 감소되었으나 2 clones은 약하게 저해되었다.
The promoter regions from chromosomal DNA of Bacillus sp. SSA3 which is responsible for fermentation of Korean traditional soy sauce, were cloned for construction of expression vector of Bacills sp. SSA3. Recombinant plasmids were constructed by insertion of HindIIl-cleaved Bacillus sp. SSA3 chromosomal DNA fragments in front of the CAT gene of pGR71 plasmid and B-galactosidase gene of pUC18 plasmid. 6 recombinant plasmids were isolated from chloramphenicol resistant E. coli JM109 clones. All these plasmids were found to have promoter activity in Bacills sp. SSA3 and E. coli JM109. When these 6 clones of Bacills sp. SSA3 were cultivated in LB agar medium supplemented with 10% NaCI. fused CAT gene expression of 4 clones was significantly decreased in common. But the others were poorly inhibited.
