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Download rpARA Recombinant mFP Expression Sequence
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Abridged Genetic Engineering Pathway (Original “A” Sequence) Recombinant Plasmid pARA-R Restriction Analysis of pARA-R Restriction fragments after digest with HindIII and BamHI BamHI HindIII araC ori ampR 4,495 bp HindIII BamHI pBAD 807 bp Restriction analysis of pARA-R (cont.) Bruce Wallace Confirmation of Restriction Prediction for restriction gel M R+ R– 10000 8000 5000 4000 3000 2000 1500 1000 500 Results for restriction gel M R+ R– Preparation of Competent Cells Add calcium chloride Heat shock Transformation of E. coli with pARA-R Competent Cells pARA-R Recombinant Plasmids Transformation of E. coli with pARA-R Lipid bilayer (inner) Peptidoglycan layer Adhesion zone Lipid bilayer (outer) Calcium ions pARA-R Growth and Selection of Transformed Bacteria P+ plates LB LB/amp P– plates No growth LB LB/amp LB/amp/ara Overnight Culture of E. coli for rfp Expression Colony isolation and culture LB/amp/ara broth RFP Production arabinose Arabinose – araC protein complex prevents DNA looping and helps to align RNA polymerase on the promoter site (PBAD). RFP (red fluorescent protein) RNA polymerase arabinose araC protein araC–protein complex Translation mRNA Transcription araC gene PBAD rfp gene RFP Purification Overnight culture Cell pellet with RFP Lysed cells Pellet RFP with cell debris binding buffer Three-dimensional Shape of RFP
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