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Engineering the pARA-R Plasmid for RFP Expression in E. Coli - Laboratory Protocol

This presentation by Bruce Wallace outlines the process of engineering the pARA-R recombinant plasmid to express red fluorescent protein (RFP) in Escherichia coli. The protocol includes detailed procedures such as restriction enzyme digestion using BamH I and Hind III, ligation of the rfp gene into the p-ARA vector, preparation of competent cells, and transformation methodologies. It further discusses the cultivation of transformed bacteria, RFP expression dynamics influenced by the araC protein, and purification techniques for the recombinant protein.

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Engineering the pARA-R Plasmid for RFP Expression in E. Coli - Laboratory Protocol

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  1. 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.3

  2. pARA-R construct Bruce Wallace Recombinant plasmid of interest BamH I pARA-R 5,302 bp PBAD-rfp 806 bp Hind III

  3. BamH I sticky end Hind III sticky end Hind III sticky end BamH I sticky end Engineering the Plasmid: ligation of rfp gene into p-ARA Bruce Wallace 3’ 5’ 3’ 5’ 5’ 3’ 5’ 5’ 3’ 3’ 3’ 5’

  4. Restriction digest of pARA-R Bruce Wallace Recombinant plasmid of interest BamH I BamH I pARA-R 5,302 bp PBAD-rfp 806 bp Hind III Hind III

  5. Restriction analysis of pARA-R Bruce Wallace Restriction fragments after digest with Hind III and BamH I BamH I Hind III 4,496 bp Hind III BamH I 806 bp

  6. 10000 8000 5000 4000 3000 2000 1500 1000 500 Restriction analysis of pARA-R Bruce Wallace Prediction for restriction gel R- R- R+ M R+ M

  7. pARA-R construct Bruce Wallace Recombinant plasmid of interest BamH I pARA-R 5,302 bp PBAD-rfp 806 bp Hind III

  8. Calcium ions Preparing competent cells for transformation Bruce Wallace Lipid bilayer (inner) Peptidoglycan layer Adhesion zone Lipid bilayer (outer)

  9. Competent Cells pARA-R Transforming Escherichia coli with pARA-R Bruce Wallace Recombinant Plasmids

  10. Calcium ions pARA-R Transforming Escherichia coli with pARA-R Bruce Wallace Lipid bilayer (inner) Peptidoglycan layer Adhesion zone Lipid bilayer (outer)

  11. Growth of transformed bacteria on various plates Bruce Wallace P+ plates LB LB/amp LB/amp/ara P- plates No growth LB LB/amp

  12. araC gene PBAD rfp gene mRNA araC protein RFP expression Bruce Wallace Transcription Translation

  13. araC protein prevents RFP transcription by causing a loop to form in the region of the fp gene r araC gene PBAD rfp gene RFP expression Bruce Wallace araC protein

  14. araC gene PBAD rfp gene RFP (red fluorescent protein) mRNA Transcription RFP expression Bruce Wallace Arabinose – araC protein complex prevents DNA looping and helps to align RNA polymerase on the promoter site (PBAD). arabinose Translation RNA polymerase arabinose – araC protein complex araC protein

  15. Bruce Wallace RFP

  16. LB/amp/ara broth Preparing an overnight culture of E. Coli for RFP expression Bruce Wallace Colony isolation and culture

  17. 24 hours later… After 5 hrs, add arabinose CC/ LB/ Amp Cloudy Clear PINK!!

  18. Purification of RFP from an overnight culture Bruce Wallace Overnight culture Cell pellet with RFP Lysed cells Pellet cell debris RFP with binding buffer

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