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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
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Order the primer to clone orf2 (pProEX™HTa vector 고려) PerformPCR 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 ->column의 배송이 지연 되는 관계로 다음주에 실행하기로 함 Overexpression of Orf2 in Mycobacterium sp. strain JC1
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 primer로 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 Mycobacteriumsmegmatis • 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 Orf2가 만들어지도록 primer 제작 • 제작된 primer로 PCR 수행 • Elute PCR product and ligate with pGEM-T easy vector (Orf1 sequencing data 분석 결과 non-specific band로 확인되어 다시 PCR 하기로 결정 , Orf2 plasmid DNA prep. 준비) • 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
Construction of bait vector for Rv3676 from Mycobacterium smegmatis • Order the primer to clone Mycobacterium smegmatis Rv3676 • 제작된 primer로 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
