Abridged Genetic Engineering Pathway (Original “A” Sequence)

Recombinant Plasmid pARA-R

Restriction fragments after digest with HindIII and BamHI 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

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

Preparation of Competent Cells Add calcium chloride Heat shock

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

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

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

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

arabinose – araC protein RFP Production RFP (red fluorescent protein) 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 mRNA Transcription araC gene PBAD rfp gene

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

Three-dimensional Shape of RFP