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BACTERIAL TRANSFORMATION and Fluorescent Proteins

BACTERIAL TRANSFORMATION and Fluorescent Proteins. Many organisms can fluoresce. Jellyfish. Amphipods Oceanexplorer.noaas.gov/explorations/05deescope/background/fluorescence. www.worldnetcams.com/sealife/cerianthus.jpg. Corals. Copepods

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BACTERIAL TRANSFORMATION and Fluorescent Proteins

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  1. BACTERIAL TRANSFORMATION and Fluorescent Proteins

  2. Many organisms can fluoresce Jellyfish Amphipods Oceanexplorer.noaas.gov/explorations/05deescope/background/fluorescence www.worldnetcams.com/sealife/cerianthus.jpg Corals Copepods Oceanexplorer.noaas.gov/explorations/05deescope/background/fluorescence www.afae.it/pages/galleria%20immagini/Invertebrati%20marini/trachy.htm www.wileywiggins.com/ 2003_05_01_blog2.html www.welc.cam.ac.uk/ ~pineslab/Projects/gfp.htm Renilla reniformis

  3. Aequorea victoria (crystal jelly) and Discovery of GFP-1960’s

  4. Scorpion- Natural Light Scorpion- UV Light Bioluminescence vs. Fluorescence Bioluminescence Fluorescence http://fireflyforest.net/firefly/2006/11/13/fluorescent-scorpion-in-uv-light/ Natural Light In the Dark A fluorescent organism absorbs light at one wavelength (UV) and a re-emits the light at a visible wavelength= color Bioluminescent organism produces its own light.

  5. Adaptations for Fluorescence • It might entice mates… • In the spider Cosmophasis umbratica, the palps of the males and females fluoresce in UV light, leading to courtship poses.

  6. Adaptations for Bioluminescence • Attract Prey • Protection • Communication

  7. Roger Tsien and Rainbow Proteins

  8. Transgenic Mice

  9. Transgenic Fish Embryos Normal Light UV Light

  10. Fly Embryo T. Megraw Marsupial Cell S.L. Kline Frog Egg Extract + DNA-coated beads R. Heald Human Cell J. Waters Worm Embryo I.M. Cheeseman Frog Egg Extract + sperm DNA A. Desai Frog Cell C.E. Walczak MITOSIS a similar process in diverse species ……...using various organisms to understand humans:

  11. E. coli

  12. Central Dogma DNA---> mRNA---> Protein---> Trait

  13. Transcription / Translation • http://www.dnai.org/a/index.html Campbell

  14. DNA RNA Protein Trait Green Fluorescent Protein • GFP Gene • found in jellyfish • engineered into bacteria GLOWING CELLS WHY SHOULD WE DO THIS? To SEE the Central Dogma in action:

  15. STARTING MATERIALS E. coli cells • sensitive to antibiotics • can’t glow • competent - able to be transformed Bacterial chromosome

  16. How are plasmids engineered? Bacterial DNA Plasmid Host DNA fragments (i.e. coral or jellyfish FP coding DNA) Ligate (paste) fragments into cut DNA plasmid Cut plasmids open with restriction enzymes + Cut genomic DNA into fragments End result: Plasmid containing FP gene

  17. AmpR GROW ON AN AGAR PLATE GFP Ara … that can GLOW! END RESULT Recombinant Bacteria…

  18. makes all transformed bacteria resistant to ampicillin Makes the bacteria glow Helps the bacteria make GFP when it eats the sugar arabinose Genes Contained on the Plasmid AmpR pGLO plasmid GFP Ara promoter

  19. Bacterial chromosome What is Transformation? Transformation is when the DNA from one organism is combined with the DNA from another organism. Plasmid Bacterial chromosome Bacteria now express fluorescent protein…

  20. Why Ampicillin? • Ampicillin inhibits cell 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

  21. Welcome to our Transformation Lab • Please put your backpack in the back of the room and quietly take a seat.

  22. Lab Safety and Sterile Technique • Wear goggles and gloves whenever you are at the lab stations • To prevent other bacteria from getting on the petri dishes (plates), don’t let the pipette tips touch anything. • Do not touch your mouth/face with your hands • Wash your hands thoroughly after the lab

  23. Today’s Plan • Pre-Lab Questions and Lab Overview • Follow Lab Procedure. While some students are conducting steps of the lab, other students will answer the questions on their lab sheet. • Clean-up and set up for the next class

  24. Clean-up/ Set Up • Spray the items in your “Waste” container with Lysol, then through them in the regular trash. • Spray your lab station and your group’s table with Lysol and wipe down the tables with paper towels. • Get 2 clear microtubes and 1 yellow microtube for the next class. • Organize your lab materials neatly on the counter.

  25. Student Roles • Person 1: Steps 1, 2, 10 • Person 2: Steps 3 and 4 • Person 3: Steps 5 and 6 • Person 4: Steps 7, 8, and 9 • Person 1/5: Steps 11, 12 • Everyone: Step 13

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