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Download rpARA Recombinant mFP Expression Sequence
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Biotechnology Lab Program Bruce Wallace pARA-R Sequence RFP Expression Sequence Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Kristi Schramm Pierce College, Woodland Hills, CA V.1.2a.2 pARA-R construct Bruce Wallace Recombinant plasmid of interest pARA-R 4720 bp rfp 702bp Engineering the Plasmid: ligation of rfp gene into p-ARA Bruce Wallace BamH I sticky end Hind III sticky end Hind III sticky end BamH I sticky end pARA-R construct Bruce Wallace Recombinant plasmid of interest pARA-R 4720 bp rfp 702bp RFP expression Bruce Wallace araC gene PBAD Transcription mRNA Translation araC protein rfp gene RFP expression Bruce Wallace araC protein prevents RFP transcription by causing a loop to form in the region of the r fp gene araC gene araC protein PBAD rfp gene RFP expression Bruce Wallace arabinose Arabinose – araC protein complex prevents DNA looping and helps to align RNA polymerase on the promoter site (PBAD). RFP (red fluorescent protein) Translation RNA polymerase arabinose araC protein araC–protein complex mRNA Transcription araC gene PBAD rfp gene Preparing competent cells for transformation Bruce Wallace Lipid bilayer (inner) Adhesion zone Peptidoglycan layer Lipid bilayer (outer) Calcium ions Transforming Escherichia coli with pARA-R Bruce Wallace Competent Cells pARA-R Recombinant Plasmids Transforming Escherichia coli with pARA-R Bruce Wallace Lipid bilayer (inner) Peptidoglycan layer Adhesion zone Lipid bilayer (outer) Calcium ions pARA-R Growth of transformed bacteria on various plates Bruce Wallace P+ plates LB LB/amp P- plates No growth LB LB/amp LB/amp/ara Preparing an overnight culture of E. Coli for RFP expression Bruce Wallace Colony isolation and culture LB/amp/ara broth Bruce Wallace RFP Purification of RFP from an overnight culture Bruce Wallace Overnight culture Cell pellet with RFP Lysed cells Pellet RFP with cell debris binding buffer
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