Overexpression of Orf2 in Mycobacterium sp. strain JC1
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Overexpression of Orf2 in Mycobacterium s p. strain JC1 Order the primer to clone orf2 (pProEX™HTa vector 고고 ) Perform PCR with produced primer using Mycobacterium sp. strain JC1 chromosomal DN A Elution PCR product and ligation with pGEM-T easy vector Transformation into E.coli DH5α Sequencing the cloned pGEM-T easy vector containing PCR product Ligate with pProEX™HTa for overexpression Transformation into E.coli DH5α Plasmid prep & Transformation into E.coli BL21 A preliminary Experiment Induction by IPTG (25℃ & 37℃) Confirm expression patterns using SDS-PAGE Induce at 37℃ for 5hr & Harvest -20℃ 고고 Protein sample extraction ->column 고 고고고 고고 고고 고고고 고고고고 고고고고고 고
description
Order the primer to clone orf2 (pProEX ™ HTa vector 고려 ) Perform PCR with produced primer using Mycobacterium sp. strain JC1 chromosomal DNA Elution PCR product and ligation with pGEM-T easy vector Transformation into E.coli DH5α - PowerPoint PPT Presentation
Transcript of Overexpression of Orf2 in Mycobacterium sp. strain JC1
Overexpression of Orf2 in Mycobacterium sp. strain JC1
Order the primer to clone orf2 (pProEX™HTa vector 고려 )
Perform PCR with produced primer using Mycobacterium sp. strain JC1 chromosomal DNA
Elution PCR product and ligation with pGEM-T easy vector
Transformation into E.coli DH5α
Sequencing the cloned pGEM-T easy vector containing PCR product
Ligate with pProEX™HTa for overexpression
Transformation into E.coli DH5α
Plasmid prep & Transformation into E.coli BL21
A preliminary Experiment Induction by IPTG (25℃ & 37℃)
Confirm expression patterns using SDS-PAGE
Induce at 37℃ for 5hr & Harvest -20℃ 보관
Protein sample extraction ->column 의 배송이 지연 되는 관계로 다음주에 실행하기로 함
Detection of protein interacted with Orf1 or Orf2 using His-tagged Orf1 or Orf2 from Mycobacterium smegmatis
His-tagged Orf1or Orf2 만들어지도록 primer 제작 제작된 primer 로 PCR 수행 Elute PCR product and ligate with pGEM-T easy vector (Orf1 PCR 재수행 primer 다시 주문 , Orf2 : Transfomation 재수행 ) Sequencing the cloned pGEM-T easy vector containing PCR product Ligate with pNBV1 for overexpression the His-tagged Orf1 or Orf2 Electroporation overexpression vector into Mycobacterium smegmatis Incubation on SMB supplemented with CO medium Purification the protein interacted with His-tagged Orf1 or Orf2 MALDI TOF analysis
Construction of bait vector for Orf1 and Orf2 from Mycobacterium smegmatis
Orf1 or Orf2 가 만들어지도록 primer 제작 제작된 primer 로 PCR 수행 Elute PCR product and ligate with pGEM-T easy vector (Orf1 sequencing data 분석 결과 non-specific band 로 확인되어
다시 PCR 하기로 결정 , Orf2 plasmid DNA prep. 준비 ) Sequencing the cloned pGEM-T easy vector containing PCR prod
uct Ligate with pAS2-1 for bait vector Transformation into E. coli Co-transformation into S. cerevisiae with prey vector
Construction of bait vector for Rv3676 from Mycobacterium smegmatis
Order the primer to clone Mycobacterium smegmatis Rv3676 제작된 primer 로 PCR 수행 Elute PCR product and ligate with pGEM-T easy vector Sequencing the cloned pGEM-T easy vector containing PCR product Ligate with pAS2-1 for bait vector Transformation into E. coli Co-transformation into S. cerevisiae with prey vector
