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? 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? Green light (Lower energy) Excited state Ground state Blue light (High energy)

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

11. GFP RFP Roger Tsien and Rainbow Proteins

13. Uses for FPs Human Cell J. Waters

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

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

16. E. coli bacterial cell Bacterial genome Plasmid Cell produces protein coded by plasmid DNA. What is Transformation? DNARNAProtein

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

18. What’s on the plasmid Amp R Ampicillin resistance gene FP gene Plasmid Mix 1 GFP BFP Grape Plasmid Mix 2 YFP Tangerine Cherry

19. How the Plasmid Gets in the Bacteria Show Video 1.Add CaCl2 2.Heat/Shock

20. Step 0: Label Plates

21. Step 1: Put CaCl 2 in Tube

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

23. Ca ++ O CH 2 O PO O O Base CH 2 O P O O O Base OH Sugar O Ca ++ Step 3: Put bacteria in CaCl 2 Positive charge of Ca 2+ ions shields negative charge of DNA phosphates

24. Step 4: Add Plasmid to (+) Tube Amp R Ampicillin resistance gene FP gene Plasmid Mix 1 GFP BFP Grape Plasmid Mix 2 YFP Tangerine Cherry

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

26. Step 6: Plate on Ampicillin cell wall Ampicillin inhibits cell wall growth. 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.

27. 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

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

29. Why Ampicillin? LB/No Amp

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

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