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pARA-R Recombinant

biotechnology lab program. pARA-R Recombinant. RFP Expression Sequence. Laboratory Protocols by:. Marty Ikkanda. Powerpoint by:. Anthony Daulo. Pierce College, Woodland Hills, CA. V.1.2.1. restriction analysis of pKAN-R and pARA. pKAN-R. 5408 bp. Bam H I. rfp 702 bp. pARA. 4058 bp.

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pARA-R Recombinant

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  1. biotechnology lab program pARA-R Recombinant RFP Expression Sequence Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Pierce College, Woodland Hills, CA V.1.2.1

  2. restriction analysis of pKAN-R and pARA pKAN-R 5408 bp BamH I rfp 702 bp pARA 4058 bp Hind III BamH I Hind III 40 bp

  3. restriction analysis of pKAN-R and pARA Restriction fragments after digest with Hind III and BamH I BamH I Hind III 4018 bp BamH I Hind III 4706 bp BamH I Hind III 702bp BamH I Hind III 40 bp

  4. K+ K- A+ A- M 10000 8000 5000 4000 3000 2000 1500 1000 500 restriction analysis of pKAN-R and pARA Prediction for restriction gel K+ K- A+ A- M

  5. BamH I sticky end Hind III sticky end Hind III sticky end BamH I sticky end ligation of pKAN-R/pARA restriction fragments

  6. ligation of pKAN-R/pARA restriction fragments Recombinant plasmid of interest pARA-R 4720 bp BamH I rfp 702bp Hind III

  7. K+ K- A+ A- L M 10000 8000 5000 4000 3000 2000 1500 1000 500 restriction analysis of pKAN-R and pARA Confirmation of restriction and ligation K+ K- A+ A- L M

  8. Calcium ions preparing competent cells for transformation Lipid bilayer (inner) Peptidoglycan layer Adhesion zone Lipid bilayer (outer)

  9. Competent Cells pARA-R transforming Escherichia coli with pARA-R Recombinant Plasmids

  10. Calcium ions pARA-R transforming Escherichia coli with pARA-R Lipid bilayer (inner) Peptidoglycan layer Adhesion zone Lipid bilayer (outer)

  11. growth of transformed bacteria on various plates P+ plates LB LB/amp LB/amp/ara P- plates No growth LB LB/amp

  12. LB/amp/ara broth preparing an overnight culture of E. Coli for RFP expression Colony isolation and culture

  13. araC gene PBAD rfp gene mRNA araC protein RFP expression Transcription Translation

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

  15. araC gene PBAD rfp gene RFP (red fluorescent protein) mRNA Transcription RFP expression 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

  16. RFP

  17. purification of RFP from an overnight culture Overnight culture Cell pellet with RFP Lysed cells Pellet cell debris RFP with binding buffer

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