1. pARA-R Recombinant RFP Expression Sequence biotechnology lab program Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Pierce College, Woodland Hills, CA V.1.2.1

2. restriction analysis of pKAN-R and pARA pKAN-R pARA BamH I Hind III BamH I Hind III rfp 702 bp 5408 bp 4058 bp 40 bp

3. restriction analysis of pKAN-R and pARA BamH I Hind III 702bp 40 bp BamH I Hind III BamH I Hind III BamH I Hind III Restriction fragments after digest with Hind III and BamH I 4018 bp 4706 bp

4. restriction analysis of pKAN-R and pARA Prediction for restriction gel M K+K-A+A- M K+K-A+A- 500 1000 1500 2000 3000 4000 5000 8000 10000

5. ligation of pKAN-R/pARA restriction fragments sticky end BamH I sticky end Hind III sticky end BamH I sticky end Hind III

6. ligation of pKAN-R/pARA restriction fragments Recombinant plasmid of interest pARA-R BamH I Hind III rfp 702bp 4720 bp

7. restriction analysis of pKAN-R and pARA M K+K-A+A- 500 1000 1500 2000 3000 4000 5000 8000 10000 Confirmation of restriction and ligation L M K+K-A+A-L

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

9. transforming Escherichia coli with pARA-R Recombinant Plasmids Competent Cells pARA-R

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

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

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

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

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

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)

16. RFP

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