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RISS 인기검색어
- 검색키워드
- 저자 : 양철학 ( Jong Sun Kim (검색결과 2 건)
- 무료
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Cloning of subtilisin Carlsberg gene from Bacillus licheniformis
김종선,이은경,양철학,Kim, Jong-Sun,Lee, Eun-Kyeong,Yang, Chul-Hak 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.2
Bacillus licheniformis로부터 썹틸리신 칼스버그 효소의 유전정보를 담고 있는 유전자를 15-mer의 합성된 올리고뉴클레오티드를 probe로 사용하여 "in-situ colony hybridization technique"으로 찾아내었다. 제한효소 Pst1으로 절단된 chromosomal DNA를 pBR322의 Pst1부위에 연결시킨 후 이것으로 대장균을 형질전환시켜 얻은 3,500여개의 콜로니를 in-situ colony hybridization technique를 써서 조사한 결과 probe에 hybridization되는 양성콜로니를 한개 얻었다. 여기에서 얻은 재조합유전자를 pS113이라 명명하였으며 제한효소로 이를 분석해 본 결과 4.6Kb 정도의 유전자가 삽입되었고 제한효소들의 절단부위는 이마 보고된 Jacobs의 결과와 대개 일치하였다. 위에서 얻은 유전자를 Bacillus에서 발현시키기 위해 제한효소 Pst1으로 절단된 pS113을 pMK4의 Pst1부위에, 연결시킨 후 이것으로 Bacillus를 형질 전환시켰다. 형질전환된 Bacillus를 카세인 플레이트에서 프로테아제의 활성을 조사해 본 결과 유난히 큰 활성을 보이는 것이 얻어졌다. The gene encoding subtilisin Carlsberg from Bacillus licheniformis has been isolated by the "in-situ colony hybridization technique" using synthetic oligonucleotide as a probe. Pst1 digested chromosomal DNA was ligated into the Pst1 site of pBR322. The ligation mixture was used to transform E. coli and the transformants were screened by the in-situ colony hybridization technique. Out of 3,500 colonies screened, one colony was hyridized to the probe. The recombinant DNA of this positive clone was named pS113. Digestion of pS113 by restriction endonucleases showed that the size of the inserted DNA was about 4.6 Kb and the restriction map was nearly like that of Jacobs'. To express the gene in Bacillus, Pst1 digested pS113 was ligated into the Pst1 site of pMK4. The ligation mixture was used to transform B. licheniformis and the transformants were assayed on casein plate for protease secretion. We obtained a clony having a distinctively large halo.
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Bacillus Licheniformis 로 부터 썹틸리신 칼스버그 유전자의 분리
김종선,이은경,양철학 ( Jong Sun Kim,Eun Kyeong Lee,Chul Hak Yang ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.2
The gene encoding subtilisin Carlsberg from Bacillus licheniformis has been isolated by the $quot;in-situ colony hybridization technique$quot; using synthetic oligonucleotide as a probe. Pstl digested chromosomal DNA was ligated into the Pstl site of pBR322. The ligation mixture was used to transform E. coli and the transformants were screened by the in-situ colony hybridization technique. Out of 3,500 colonies screened, one colony was hyridized to the probe. The recombinant DNA of this positive clone was named pS113. Digestion of pS113 by restriction endonucleases showed that the size of the inserted DNA was about 4.6 Kb and the restriction map was nearly like that of Jacobs`. To express the gene in Bacillus, Pstl digested pS113 was ligated into the Pstl site of pMK4. The ligation mixture was used to transform B. licheniformis and the transformants were assayed on casein plate for protease secretion. We obtained a colony having a distinctively large halo.
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