1. pARA-R Sequence RFP Expression Sequence Biotechnology Lab Program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Kristi Schramm Pierce College, Woodland Hills, CA V.1.2a.3 Bruce Wallace

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

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

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

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

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

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

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

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

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

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

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

13. RFP expression araC generfp geneP BAD Transcription mRNA Translation araC protein Bruce Wallace

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

15. RFP expression 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) Bruce Wallace

16. RFP Bruce Wallace

17. RFP Bruce Wallace

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