Abridged Genetic Engineering Pathway (Original “A” Sequence)

1. Abridged Genetic Engineering Pathway (Original “A” Sequence)

2. Recombinant Plasmid pARA-R

3. Restriction Analysis of pARA-R Restriction fragments after digest with HindIII and BamHI BamHI HindIII araC ori ampR 4,495 bp HindIII BamHI pBAD 807 bp

4. 10000 8000 5000 4000 3000 2000 1500 1000 500 Restriction analysis of pARA-R (cont.) Confirmation of Restriction Bruce Wallace Prediction for restriction gel Results for restriction gel M R+ R– M R+ R–

5. Preparation of Competent Cells Add calcium chloride Heat shock

6. Competent Cells pARA-R Transformation of E. coli with pARA-R Recombinant Plasmids

7. Calcium ions pARA-R Transformation of E. coli with pARA-R Lipid bilayer (inner) Peptidoglycan layer Adhesion zone Lipid bilayer (outer)

8. Growth and Selection of Transformed Bacteria P+ plates LB LB/amp LB/amp/ara P– plates No growth LB LB/amp

9. LB/amp/ara broth Overnight Culture of E. coli for rfp Expression Colony isolation and culture

10. araC gene PBAD rfp gene RFP (red fluorescent protein) mRNA Transcription RFP Production 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

11. RFP Purification Overnight culture Cell pellet with RFP Lysed cells Pellet cell debris RFP with binding buffer

12. Three-dimensional Shape of RFP