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pARA-R Sequence

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. pARA-R construct. Bruce Wallace. Recombinant plasmid of interest.

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pARA-R Sequence

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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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