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pARA-R Sequence RFP Expression Sequence Biotechnology Lab Program

Published byJosé Antonio Alcaraz Modified over 5 years ago

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Presentation on theme: "pARA-R Sequence RFP Expression Sequence Biotechnology Lab Program"— Presentation transcript:

1 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

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

3 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

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

5

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

7 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

8 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

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

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

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

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

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

14 Bruce Wallace RFP

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

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