Bacterial Transformation Lab
- 1. Warmup Draw 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? A rod shaped bacterium found in our lower intestines What is fluorescence? 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 E. coli bacterial cell Bacterial genome Bacterial genome
- 10. DNA Transcription RNA Translation Protein E. coli bacterial cell E. coli bacterial cell Bacterial genome Bacterial genome I think we should alter the ________________ because _____________ DNA RNA Protein
- 11. How We Make E.coli Glow Plasmid Uptake of foreign DNA, often a circular plasmid Allow bacteria to grow for 1-3 days DNA RNA Protein Bacteria now express cloned fluorescent 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. What is on the Plasmid? E. coli bacterial cell FP Bacterial genome promoter Multiple Cloning Site Origin (MCS) Stop AmpR (selectable marker)
- 14. What’s on the plasmid Plasmid Mix 1 Plasmid Mix 2 GFP Cherry FP gene BFP Tangerine Grape YFP AmpR Ampicillin resistance gene
- 15. How We Make E.coli Glow Plasmid Uptake of foreign DNA, often a circular plasmid Allow bacteria to grow for 1-3 days DNA RNA Protein Bacteria now express cloned fluorescent protein…
- 16. How the Plasmid Gets in the Bacteria Show Video 1. Add CaCl2 2. Heat/Shock
- 17. Why Ampicillin? LB/AMP + LB/AMP -
- 19. Why Care? Frog Cell C.E. Walczak Worm Embryo I.M. Cheeseman
Editor's Notes
- #10: How do we make a protein?If I wanted to make a new protein then what part of the central dogma should I change?
- #11: Think for about what part of this process you will change and why?For example, Maybe we should change RNA because ______________Have students raise their hands and call on them by order numberIf I wanted to make a new protein then what part of the central dogma should I change?
- #14: Draw Diagram of Plasmid with students and explain the parts