Bacterial Transformation Lab

1. Warmup D raw or explain in words in the simplest way you can the process of Protein synthesis (aka Central Dogma aka Transcription/Translation) Think of an awesome trait you would want to have. For example, synesthesia, being taller, curly hair etc. Explain how would you use the process of protein synthesis to get that trait? For Quanina’s Graduate Studies: Have you taken a comprehensive health course in your educational career? If so, what grade were you in and what did it cover (ex: sex ed , family planning, etc. )? What school were you at when you took this course?

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

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

4. The Purpose of the Labs The purpose of this labs are to “transform” (change) a normal E. coli bacterial cell into an E.coli bacterial cell that produces fluorescent protein (FP ) using what we know about the central dogma. Finally, we will isolate the FP .

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

6. What is Fluorescence? Fluorescence : the emission of light by something that has absorbed light at a different wavelength

7. NOTE: FLUORESCENCE IS NOT LUMINESCENCE Fluorescence Bioluminescence

8. Why do we care? Because it’s cool! Frog Cell C.E. Walczak

9. How will we make E. coli bacteria produce the red fluorescent protein? E. coli bacterial cell Bacterial genome Bacterial genome E. coli bacterial cell

10. E. coli bacterial cell Bacterial genome Bacterial genome E. coli bacterial cell I think we should alter the ________________ because _____________ DNA RNA Protein   Transcription   Translation Protein RNA DNA

11. 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

12. Characteristics of a Plasmid : Circular Piece of DNA Occurs Naturally Can be engineered (made) in the lab and then inserted into bacteria to make a desired protein . Can contain antibiotic resistance genes and/or other genes Not incorporated to the bacterial genome but can replicate on its own

13. E. coli bacterial cell Bacterial genome Origin Amp R (selectable marker ) FP Stop promoter Multiple Cloning Site (MCS) What is on the Plasmid?

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

15. 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

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

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

18. Why Ampicillin? LB/No Amp

19. Why Care? Frog Cell C.E. Walczak Worm Embryo I.M. Cheeseman