Biotechnology Lab Program Bruce Wallace pKAN-R Sequence Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Pierce College, Woodland Hills, CA V.1.2.5
Download ReportTranscript Biotechnology Lab Program Bruce Wallace pKAN-R Sequence Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Pierce College, Woodland Hills, CA V.1.2.5
Biotechnology Lab Program Bruce Wallace pKAN-R Sequence Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Pierce College, Woodland Hills, CA V.1.2.5 Restriction analysis of pKAN-R and pARA Bruce Wallace pKAN-R BamHI 5,512 bp PBAD-rfp 807 bp pARA 4,872 bp 377 bp Restriction analysis of pKAN-R and pARA Bruce Wallace Restriction fragments after digest with Hind III and BamH I BamH I Hind III 4,705 bp BamH I Hind III 4,495 bp Hind III BamH I 807 bp Hind III BamH I 377 bp Restriction analysis of pKAN-R and pARA Bruce Wallace Prediction for restriction gel M A+ A- K+ K- 10000 8000 5000 4000 3000 2000 1500 1000 500 M A+ A- K+ K- Ligation of pKAN-R/pARA restriction fragments Bruce Wallace BamH I sticky end 3’ 5’ 3’ 5’ sticky end 5’ 5’ 3’ 3’ Hind III BamH I sticky end Hind III sticky end 3’ 5’ 3’ 5’ Ligation of pKAN-R/pARA restriction fragments Bruce Wallace Recombinant plasmid of interest pARA-R 5,302 bp PBAD-rfp 807 bp Restriction analysis of pKAN-R and pARA Bruce Wallace Confirmation of restriction and ligation M A+ A- K+ K- L 10000 8000 5000 4000 3000 2000 1500 1000 500 M A+ A- K+ K- L 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) Peptidoglycan layer Adhesion zone Lipid bilayer (outer) Calcium ions pARA-R Growth of transformed bacteria on various plates Bruce Wallace P+ plates LB LB/amp P- plates No growth LB LB/amp LB/amp/ara Preparing an overnight culture of E. Coli for RFP expression Bruce Wallace Colony isolation and culture LB/amp/ara broth RFP expression Bruce Wallace araC gene PBAD Transcription mRNA Translation araC protein rfp gene RFP expression Bruce Wallace araC protein prevents RFP transcription by causing a loop to form in the region of the r fp gene araC gene araC protein PBAD rfp gene RFP expression Bruce Wallace arabinose Arabinose – araC protein complex prevents DNA looping and helps to align RNA polymerase on the promoter site (PBAD). RFP (red fluorescent protein) Translation RNA polymerase arabinose araC protein araC–protein complex mRNA Transcription araC gene PBAD rfp gene Bruce Wallace RFP Purification of RFP from an overnight culture Bruce Wallace Overnight culture Cell pellet with RFP Lysed cells Pellet RFP with cell debris binding buffer