Bacterial Transformation Lab

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  • How do we make a protein?If I wanted to make a new protein then what part of the central dogma should I change?
  • 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?
  • Draw Diagram of Plasmid with students and explain the parts
  • Bacterial Transformation Lab

    1. 1. WarmupDraw or explain in words in the simplest way you can theprocess of Protein synthesis (aka Central Dogma akaTranscription/Translation)Think of an awesome trait you would want to have. Forexample, synesthesia, being taller, curly hair etc. Explainhow would you use the process of protein synthesis to getthat trait?For Quanina’s Graduate Studies: Have you taken acomprehensive 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 atwhen you took this course?
    2. 2. Bacterial Transformation Student TrainingBy Quanina Quan and UCSD ScienceBridge
    3. 3. ObjectiveDemonstrate that changes in genotype causes changesin phenotype by transforming E.coli into fluorescentE.coli.
    4. 4. The Purpose of the LabsThe purpose of this labs are to “transform” (change) anormal E. coli bacterial cell into an E.coli bacterial cellthat produces fluorescent protein (FP) using what weknow about the central dogma. Finally, we will isolatethe FP.
    5. 5. BackgroundWhat is E.coli? A rod shaped bacterium found in our lower intestinesWhat is fluorescence? Absorbs light at one wavelength (UV) and re-emits light at a visible wavelength (color)
    6. 6. What is Fluorescence?Fluorescence: the emission of light by something thathas absorbed light at a different wavelength
    7. 7. NOTE: FLUORESCENCE IS NOT LUMINESCENCEFluorescence Bioluminescence
    8. 8. Why do we care? Because it’s cool! Frog Cell C.E. Walczak
    9. 9. How will we make E. coli bacteria produce the red fluorescent protein?E. coli bacterial cell E. coli bacterial cell Bacterial genomeBacterial genome
    10. 10. DNA Transcription RNA Translation Protein E. coli bacterial cell E. coli bacterial cell Bacterial genome Bacterial genomeI think we should alter the ________________ because _____________ DNA RNA Protein
    11. 11. How We Make E.coli Glow Plasmid Uptake of foreign DNA, often a circular plasmid Allow bacteria to grow for 1-3 daysDNA  RNA Protein Bacteria now express cloned fluorescent protein…
    12. 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. 13. What is on the Plasmid? E. coli bacterial cell FP Bacterial genome promoter Multiple Cloning SiteOrigin (MCS) Stop AmpR (selectable marker)
    14. 14. What’s on the plasmidPlasmid Mix 1 Plasmid Mix 2 GFP Cherry FP gene BFP Tangerine Grape YFP AmpR Ampicillin resistance gene
    15. 15. How We Make E.coli Glow Plasmid Uptake of foreign DNA, often a circular plasmid Allow bacteria to grow for 1-3 daysDNA  RNA Protein Bacteria now express cloned fluorescent protein…
    16. 16. How the Plasmid Gets in the BacteriaShow Video1. Add CaCl22. Heat/Shock
    17. 17. Why Ampicillin? LB/AMP + LB/AMP -
    18. 18. Why Ampicillin?LB/No Amp
    19. 19. Why Care?Frog CellC.E. Walczak Worm Embryo I.M. Cheeseman

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