1. BACTERIAL TRANSFORMATION and Fluorescent Proteins

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

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

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

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

6. Adaptations for Bioluminescence Protection Attract Prey Communication

7. Roger Tsien and Rainbow Proteins

8. Transgenic Mice

9. Transgenic Fish Embryos Normal LightUV Light

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

11. E. coli

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

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

14. WHY SHOULD WE DO THIS? To SEE the Central Dogma in action: DNARNAProteinTrait GFP Gene found in jellyfish engineered into bacteria Green Fluorescen t Protein GLOWING CELLS

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

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

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

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

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

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

22. Welcome to our Transformation Lab Please take out your pGLO lab packet and a pen or pencil put your backpack in the back of the room.

23. Today’s Plan 1.Pre-Lab Questions and Lab Overview 2.Follow Lab Procedure. While some students are conducting steps of the lab, other students will answer the questions on their lab sheet. 3.Clean-up and set up for the next class

24. pGLO Supplies

25. What is a starter plate? E. coli starter plate This plate has the bacteria we will use growing in a luria broth (LB) agar plate. These bacteria are normal (have NOT been transformed)

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

27. What is in each of the 4 plates?

28. Lab Safety and Sterile Technique Wear goggles, aprons 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

29. Clean-up/ Set Up 1.Spray the items in your “Waste” container with Lysol, then throw them in the regular trash. 2.Spray your lab station and your group’s table with Lysol and wipe down the tables with paper towels. 3.Set up your lab station for the next class.

30. pGLO Supplies

31. Analysis Questions 1.List the function of each of the following materials used in the lab: LB agar, ampicillin, arabinose, E. coli, petri dish, Amp R gene, Ara C gene, GFP gene, plasmid. 2.Draw each of your petri dishes and describe what you observed in each plate. Which ones grew and which ones had fluorescent bacteria? 3.Explain why your lab group experienced the results it did in each plate. Was this what you expected? What could have led to differences? 4.How could scientists use fluorescent proteins to solve real world problems?