1.  Tuesday May 3 Get out your journal open to next open page and have writing utensil Big idea!!: DNA to mRNA to Protein to Trait Question: If we make a change to the DNA in an organism, what will happen? By the end of class today, I should be able to: 1. Understand some basics of genetically modifying an organism (GMO!) 2. Summarize & Flow Chart the steps in a genetic modification process

2. Central Dogma of Molecular Biology If we put in new DNA into an organism– what will happen to the organism? DNA RNA ProteinTraitBackground:

3. This Little Light of Mine: Aequorea victoria: Source of “glowing gene” for this experiment Transform bacteria with a jellyfish gene to make them glow AP Lab 8 Bacterial Transformation

4. Jellyfish Gene put into Other Critters

5. Agenda  Prep pFLO lab  Begin Genetic Engineering Lab

7. Gene Plasmid Transformation-process of giving another organism a new gene. Plasmids can “carry” multiple genes.

8. Genes-R-Us  Produce genes and their protein products  This project produces proteins that produce a color and fluoresce (glow).  Insert the pFLO plasmid into a harmless strain of the E. coli bacteria  The E. coli will be our “factory” that produces the color protein.  It also includes an ampicillin resistant gene.

9. How do antibiotics work and what is antibiotic resistance?  Fungus and bacteria compete for same resources  Fungus make ampicillin to kill bacteria (and we use it for medicine)  Ampicillin binds to enzyme that builds bacterial cell walls – kills bacteria  Ampicillin resistant bacteria have an enzyme that destroys ampicillin  Battle is ON!!!

10. Genes-R-Us New Lab Notebook Heading: Genes-R-Us Your objective (s) 1. Learn how to genetically modify the genes in (GMO) a bacteria so that it has a new trait. 2. Suggest possible improvements to the bacterial transformation process

11. Genes-R-Us Hypothesis: If we insert a gene for a glowing colored protein, then…. E.coli without pFLO (control) E.coli with pFLO LB LB/Amp E. coli colonies are usually white. E. coli with pFLO will be colored and fluoresce under UV light.

12. Genes-R-Us Procedures 1. Remove excess bacteria from table top by  cleaning with disinfectant (ETOH)  wash hands!

13. Genes-R-Us 2. Get 2 mictrotubes with 250 μ l cold CaCl 2. 3. Label with group # and one with “C” for Control and the other with “pFLO.” Put on ice. CpFLO CaCl 2 solution Why CaCl2? Would a (-) charged molecule like DNA be able to get through the cell membrane? Ca++ neutralizes DNA

14. Genes-R-Us 4. Use a sterile toothpick to gently scrape up a large colony of bacteria from the stock plate. Add to C tube. SPIN and MIX! Repeat with new sterile toothpick into pFLO tube. Return to ice. CpFLO

15. Genes-R-Us 4. Use a sterile micropipette tip to transfer 10 μ l of pFLO plasmid (stock tube at front) to pFLO tube. 5. Add 10 μ l sterile water to tube C. 6. Mix by gently tapping tube. Let rest on ice for 15 minutes! Read next steps carefully. Timing is essential! CpFLO 15 minutes

16. Genes-R-Us 5. Label your four plates as indicated. Also include group #/name & class period Read next steps carefully. Timing is essential! c c

17. Genes-R-Us 6. Take ice container with tubes to the 42° C water bath. Place tubes in water bath for exactly 90 seconds. Immediately return to ice for at least 2 minutes. 90 seconds

18. Genes-R-Us 7. After 2 minutes on ice, add 250 μ l Luria broth (LB) to each tube, using sterile micropipette tip for each one. C pFLO

19. Genes-R-Us 8. Incubate tubes in incubator for 15-30 minutes.

20. Genes-R-Us 9. Set micropipette to 100 μ l. Use a sterile tip & spreader to plate each tube! Pipette 100 μ l from tube C onto the Control LB plate. Evenly spread cells over surface of plate using sterile cell spreader. Cover. Repeat process for other C and both pFLO plates. c c

21. Genes-R-Us 10. Allow 5 minutes for plates surfaces to absorb cells. Place your four plates upside-down, tape to secure and label with group period (don’t mummify!) and put into the 37° incubator.

22. Genes-R-Us Data: E.coli without pFLO (control) E.coli with pFLO LB LB/Amp

23. Results!