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E . coli 의 Tryptophan 오페론 재조합 프라스미드의 불안정성에 관한 연구
김성훈,최영철,유두영,이세영 ( Sung Hoon Kim,Young Chul Choi,Dewey D . Y . Ryu,Se Yong Lee ) 생화학분자생물학회 1982 BMB Reports Vol.15 No.4
pBR322 and trp operon from φ80 trp or ColEl-trp was covalently joined by T4 ligase and introduced into E. coli JA221 r_k^-m_k^+ ΔtrpE5 recA, and the recombinant was selected on the VB minimal plate containing ampicillin (50 ㎍/㎖) and tetracycline (10 ㎍/㎖), The growth rate of transformants obtained was very low and they lost their trp^+ marker very rapidly. In MV12/ pVH5, when host repression system of the tryptophan biosynthesis was partially derepressed with indoleacrylic acid, the enzyme activity of trp operon was increased but the stability of pVH5 was inversely decreased with the concentration of indoleacrylic acid added. In the glucose limited continuous culture of this strain, trp variant began to appear after about 30 generations and the trp fraction increased gradually. Trp cells isolated from cultures grown under various conditions have lost their entire plasmids or arbored modified plasmids which increased or decreased in molecular weight compared to original pVH5.
Instability of Trp Operon Recombinant Plasmids in E. coli
김성훈,최영철,유두영,이세영,Kim, Sung-Hoon,Choi, Young-Chul,Ryu, Dewey D.Y.,Lee, Se-Yong 생화학분자생물학회 1982 한국생화학회지 Vol.15 No.4
Plasmid pBR322와 ${\phi}80h$-pttrp190 혹은 ColEl-trp으로 부터 분리된 tryptophan operon을 T4 ligase에 의해 결합시켜 E. coil JA221 $r_k{^-}m_k{^+}$ recA ${\Delta}trpE5$에 도입해 ampicillin(50 ${\mu}g/ml$)과 tetracycline (10 ${\mu}g/ml$)을 함유한 Vogel Bonner minimal plate에서 그 recombinant를 선별하였다. 그러나 얻어진 transformant의 성장속도는 매우 느렸으며 그들의 $trp^+$ marker를 매우 빠른 속도로 상실하였다. MV12/pVH5 strain에서 tryptophan 생합성의 host repression sysyem이 indoleacrylic acid에 의해 부분적 derepression을 일으켰을 때 tryptophan operon의 효소 활성은, 가해준 indoleacrylie acid의 농도에 따라 증가하는 반면 pVH5의 안정성은 역으로 감소하였다. 또 이 strain을 glucose를 제한한 배지에서 연속 배양하였을 때 약 30세대가 지난 후부터 $trp^-$ variant가 나타나기 시작했으며 $trp^-$ 부분은 점차적으로 증가하였다. 그밖에 여러 다른 배양조건들로 부터 분리된 $trp^-$ 변종들은 원래의 pVH5에 비해 그 분자량이 증가되거나 감소된 형태의 plasmid를 가지고 있거나 plasmid 전체를 상실하였다. pBR322 and trp operon from ${\phi}80$ trp or ColEl-trp was covalently joined by T4 ligase and introduced into E. coli JA221 $r_k{^-}m_k{^+}$ ${\Delta}trpE5$recA, and the recombinant was selected on the VB minimal plate containing ampicillin (50 ${\mu}g/ml$) and tetracycline (10${\mu}g/ml$), The growth rate of transformants obtained was very low and they lost their $trp^+$ marker very rapidly. In MV12/ pVH5, when host repression system of the tryptophan biosynthesis was partially derepressed with indoleacrylic acid, the enzyme activity of trp operon was increased but the stability of pVH5 was inversely decreased with the concentration of indoleacrylic acid added. In the glucose limited continuous culture of this strain, $trp^-$ variant began to appear after about 30 generations and the $trp^-$ fraction increased gradually. $Trp^-$ cells isolated from cultures grown under various conditions have lost their entire plasmids or arbored modified plasmids which increased or decreased in molecular weight compared to original pVH5.