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Published byKerry Ellis Modified over 9 years ago
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Abridged Genetic Engineering Pathway (Original “A” Sequence)
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Recombinant Plasmid pARA-R
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Restriction Analysis of pARA-R Restriction fragments after digest with HindIII and BamHI 807 bp BamHIHindIII BamHIHindIII 4,495 bp pBAD ori ampR araC
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Restriction analysis of pARA-R (cont.) Prediction for restriction gel MR+R– 500 1000 1500 2000 3000 4000 5000 8000 10000 MR+R– Bruce Wallace Results for restriction gel Confirmation of Restriction
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Add calcium chloride Heat shock Preparation of Competent Cells
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Recombinant Plasmids Competent Cells pARA-R Transformation of E. coli with pARA-R
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Calcium ions pARA-R Adhesion zone Lipid bilayer (inner) Lipid bilayer (outer) Peptidoglycan layer Transformation of E. coli with pARA-R
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Growth and Selection of Transformed Bacteria P+ plates P– plates LBLB/ampLB/amp/ara LBLB/amp No growth
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Colony isolation and culture LB/amp/ara broth Overnight Culture of E. coli for rfp Expression
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RFP Production araC protein arabinose araC generfp geneP BAD arabinose – araC protein complex RNA polymerase Arabinose – araC protein complex prevents DNA looping and helps to align RNA polymerase on the promoter site (P BAD ). mRNA Transcription Translation RFP (red fluorescent protein)
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RFP Purification Overnight culture Cell pellet with RFP Lysed cells Pellet cell debris RFP with binding buffer
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Three-dimensional Shape of RFP
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