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Transformation of an Alkalin Protease Overproducer, Vibrio metschnikovii Strain RH530, and Improvement of Plasmid Stability by the par Locus
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  • Transformation of an Alkalin Protease Overproducer, Vibrio metschnikovii Strain RH530, and Improvement of Plasmid Stability by the par Locus
  • Transformation of an Alkalin Protease Overproducer, Vibrio metschnikovii Strain RH530, and Improvement of Plasmid Stability by the par Locus
저자명
Chung. So-Sun,Shin. Yong-Uk,Kim. Hee-Jin,Jin. Ghee-Hong,Lee. Hyune-Hwan
간행물명
Journal of microbiology and biotechnology
권/호정보
2001년|11권 2호|pp.222-228 (7 pages)
발행정보
한국미생물생명공학회
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정기간행물|ENG|
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이 논문은 한국과학기술정보연구원과 논문 연계를 통해 무료로 제공되는 원문입니다.
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기타언어초록

Vibrio metschnikovii strain RH530 is a non-pathogenic, industrially-important alkaline protease producer which has been isolated from wastewater. In this paper, we report on the transformation of this strain by using the method of electroporation. A field strength of $7.5;kVcm^{-1}$ and $25;{mu}F$, and using a 0.2-cm cuvette, appeared to be the optimal conditions for electroporation of the cells with the recombinant pSBCm plasmid carrying the vapK alkaline protease gene and the ColE1 replicon. Cells were subjected to osmotic shock in order to remove extracelluar DNase, and adding 200 mM of sucrose to electroporation buffer cells showed an increased transformation efficiency. Maximum efficiency of transformation was obtained at an early exponential growth phase. Using all of the conditions mentioned above, we routinely obtained a transformation efficiency of more than $10^4{({mu}g;plasmid;DNA)}^{-1}$. The stability of the plasmid pSBCm in V. metschnikovii RH530 was 25% after 18h of growth (27 generations) in the medium without antibiotic selection. The insertion of the par locus to the pSBCm increased the stability of the plasmid up to 42% without selective pressure. The increase in plasmid stability was accompanied by the increase in the productivity of alkaline protease in the recombinant V. metschnikovii strain RH530. Determining optimal conditions for the transformation of the industrially-important, nonpathogenic Vibrio strain, and the improvement of plasmid stability by introducing the par locus into the high copy number plasmid vector, will allow the development of procedures involved in the genetic manipulation of this strain, particularly for its use in the production of industrial enzymes such as alkaline protease.