pARA-R Sequence

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.2

2. pARA-R construct Bruce Wallace Recombinant plasmid of interest pARA-R 4720 bp BamH I rfp 702bp 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

4. pARA-R construct Bruce Wallace Recombinant plasmid of interest pARA-R 4720 bp BamH I rfp 702bp Hind III

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

7. 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

8. 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

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

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

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

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

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

14. Bruce Wallace RFP

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