pARA-R Sequence RFP Expression Sequence 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

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

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

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

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

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

arabinose – araC protein RFP expression Bruce Wallace 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 rfp gene PBAD

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

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

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

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

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

Bruce Wallace RFP

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