Way to Glow! Bacterial Transformation

1. Way to Glow! Bacterial Transformation Transformation of E. coli with pFluoroGreen

2. What is our goal? • To express green fluorescent protein (GFP) into transformed bacterial cells

3. Laboratory Safety • Gloves • Goggles • Lab coat • Non-pathogenic strain of E. coli

4. Transformation of E. coli • Label one microcentrifuge tube ‘- DNA.’ • Label 2nd microcentrifuge tube ‘+ DNA.’ Label on top of the lid

5. How to use a micropipette • White Round Knob: First Stop - to measure the desired amount of liquid Second Stop - to dispense all the of liquid and air in the pipette tip • Tip Ejection Button: - to eject pipette tip into waste container

6. Using a micropipette CaCl2 3. Using a F250 micropipette, add 250 µl of ice cold CaCl2 solution to each tube. 250 µl 250 µl - DNA + DNA

7. Pick your colony • Pick colonies from source plate of E. coli cells 5. Use a sterile loop to transfer several (3-4 colonies) from the source plate to +DNA and– DNA tube 6. Between your fingers, twist the loop vigorously in the cold CaCl2 to dislodge the cells

8. Adding the plasmid 7. To the ‘+ DNA’ tube add 10 µlpFG. Add 10 µl pFG + DNA

9. Incubate on ice 8. Incubate tubes on ice for 10 min.

10. Bacterial DNA Bacterial cell Plasmid DNA Genomic DNA

11. What is a plasmid? Circular piece of DNA Contain gene for GFP(Green Fluorescent Protein) Also has gene for Ampicillin resistance (AmpR) “ori” is the site where replication begins

12. What is transformation? Uptake of foreign DNA (plasmid) by the bacterial cell Gives the bacterium a new genetic trait! GFP Ampicillin Resistance protein

13. Bioluminescence • Biochemical process • Living organism gives off light • Bacteria, fungi, worms, jellyfish, insects, marine fish

14. Jellyfish GFP gene • Bioluminescent jelly fish Aquorea victoria: natural host of green fluorescent protein (GFP) • GFP is produced in special photogenic cells in the base of the jellyfish umbrella

15. Fluorescent proteins • The jelly fish Aquorea victoria, produces a photoprotein aequorin which interacts with the GFP to produce green light • In the transformed E. coli cells, the GFP fluoresces green only when exposed to long wave UV light

16. Bacterial Transformation: The real world • Bioremediation • Agriculture • Medicine

17. Heat shock • Place both tubes at • 42°Cfor90 seconds. • 10. Return tubes immediately to ice • and incubate for • 2 minutes.

18. LB 250 µl 250 µl + DNA - DNA Bacteria recovery 11. Using a sterile pipette, add 250 µl of Luria Broth (LB) to each tube. Change tips. 12. Incubate cells for 20 minutes in 37°C dry incubator for a recovery period.

19. Label agar plates Amp/-DNA Amp/+DNA Ref # ― Grp # Ref # ― Grp # 13. Label two plates Amp/-DNA 14. Label the other two plates Amp/+DNA

20. What happens in each transformation step? • Transformation solution = CaCl2 • Positive charge of Ca2+ ions neutralizes • cell membrane phospholipids • negative charge of phosphate groups in DNA • The Cl- ions move into the cell taking H2O molecules with them, which causes the cell to swell up.

21. What happens in the heat shock step? • Creates a small convection current that causes the plasmid to get pushed into the cell • Duration of heat shock is critical to experimental success

22. What happens in thebacterial recovery step? What is LB? • Luria-Bertani (LB) broth • Made of yeast, enzymatic digest of meat products • Medium that contains nutrients for bacterial growth and gene expression • Carbohydrates • Amino acids • Nucleotides • Salts • Vitamins • Allows cells to grow

23. Expected results -DNA +DNA CaCl2 E. coli LB CaCl2 E. coli pFG LB Growth Green glowing colonies No growth No glowing colonies By counting the number of colonies, you can then calculate the transformation efficiency: Number of transformants X final vol. at recovery = Number of µg of plasmid DNA vol. plated transformants per µg Amp/-DNA Amp/+DNA

24. How to plate cells 15. Remove the tubes from the dry bath incubator 16. Plate the cells as follows: -DNA +DNA 250 µl 250 µl 250 µl 250 µl Amp/-DNA Amp/-DNA Amp/+DNA Amp/+DNA

25. How to spread the cells 17. Spread the cells with a sterile inoculating loop 18. Cover both plates and allow the liquid to be absorbed Spread cells in 1 direction Same plate: spread cells 90° to first direction

26. Preparing plates for incubation 19. Stack your group’s set of plates on top of one another and tape them together • Keep plates in upright position 20. Wait for 24 hours! • Plates will incubate at 37°C in a bacterial incubation oven

27. Clean up • Bring the waste bins and plates to the front • Put the pipettes, tips and microcentrifuge tube box into the drawers (refer to the pic at the back of the drawer) • Put the other things into the basket • Wipe the table with Virox wipes • Now you can remove your gloves and goggles • Sanitize your hands with purell. Wash your hands with soap after you leave • Return the lab coats

28. Thank you and goodbye!