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Announcements

Announcements. New Weekly Schedule Observer on March 6 and 13. Measuring Zone of Inhibition. Bacterial Transformation. Student Training By Quanina Quan and UCSD ScienceBridge. Objective.

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Announcements

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  1. Announcements • New Weekly Schedule • Observer on March 6 and 13

  2. Measuring Zone of Inhibition

  3. Bacterial Transformation Student Training By Quanina Quan and UCSD ScienceBridge

  4. Objective Demonstrate that changes in genotype causes changes in phenotype by transforming E.coli into fluorescent E.coli.

  5. Background What is E.coli? What is fluorescence? A rod shaped bacterium found in our lower intestines A substance that absorbs light at one wavelength (UV) and re-emits light at a visible wavelength (color)

  6. Background: What is GFP? Green Fluorescent Protein Chromophore

  7. How does fluorescence work? Excited state Blue light(High energy) Green light(Lower energy) Ground state

  8. NOTE: FLUORESCENCE IS NOT LUMINESCENCE Fluorescence: Absorbs light at one wave length and emits it at another Bioluminescence: Produces its own light

  9. A Metaphor Fluorescence: Absorbs light at one wave length and emits it at another Bioluminescence: Produces its own light

  10. Fluorescent Organisms Fluorescence: Absorbs light at one wave length and emits it at another

  11. Roger Tsien and Rainbow Proteins GFP RFP

  12. Human Cell J. Waters Uses for FPs

  13. FAQs • Can I make myself or someone I know glow? • Can I get a glowing pet? • Can we turn it on or off?

  14. What is a plasmid? • A small circular piece of DNA • Naturally occurring • Can be altered in lab to express protein of interest

  15. What is Transformation? E. coli bacterial cell Bacterial genome DNA RNA Protein Plasmid Cell produces proteincoded by plasmid DNA.

  16. How We Make E.coli Glow Uptake of foreign DNA, often a circular plasmid Plasmid Allow bacteria to grow for 1-3 days DNA  RNA Protein Bacteria now express cloned fluorescent protein…

  17. What’s on the plasmid Plasmid Mix 1 Plasmid Mix 2 GFP Cherry FP gene BFP Tangerine Grape YFP AmpR Ampicillinresistancegene

  18. How the Plasmid Gets in the Bacteria Show Video Add CaCl2 Heat/Shock

  19. Step 0: Label Plates

  20. Step 1: Put CaCl2 in Tube

  21. Step 2: Collect bacterial colonies “Like scraping whipped cream off of Jello.”

  22. Ca++ Step 3: Put bacteria in CaCl2 O Ca++ O P O Base O O CH2 Sugar O Ca++ O O P Base O O CH2 Sugar OH Positive charge of Ca2+ ionsshields negative charge ofDNA phosphates

  23. Step 4: Add Plasmid to (+) Tube Plasmid Mix 1 Plasmid Mix 2 GFP Cherry FP gene BFP Tangerine Grape YFP AmpR Ampicillinresistance gene

  24. Step 5: Heat Shock! • Incubate on ice to slow fluid cell membrane • Heat-shock increases permeability of membranes Leave in heat 45 seconds!!! - Too short, and bacteria won't let in plasmid. - Too long, and the bacteria will die. ICE– HEAT – ICE

  25. Step 6: Plate on Ampicillin • Ampicillin inhibits cell wallgrowth. • Only cells that can inactivate the ampicillin around them will grow. • Ampicillin resistance is tied to (expressed with) the fluorescent protein gene. • Ampicillin is a selection mechanism that only allows transformed bacteria to grow on the plate.

  26. We have two controls LB only (-) = Control 1: check for viable cells LB amp (-) = Control 2: check for ampicillin function LB amp (+) = Experimental plate: grow transformants

  27. Why Ampicillin? LB/AMP + LB/AMP -

  28. Why Ampicillin? LB/No Amp

  29. Tricky Parts of Lab Labeling Plates Getting Bacteria Pipetting the right amount Plating

  30. FAQ • Why don’t I see anything glowing right now?

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