BACTERIAL TRANSFORMATION TRAINING

1. BACTERIAL TRANSFORMATION TRAINING

2. An Elegant Way to Study Developmental Neurobiology (with a little help from GFP) Gloriana Gallegos Trujillo Jin Lab

3. We can visualize the C. elegansnervous system using fluorescent proteins nose “brain” tail Bessereau Lab

4. Why do we useGreen Fluorescent Protein? • To find out the specific cells where a protein is made • To find out specific times during development proteins are made • To find out what subcellular location the protein is in: is it in the nucleus? In the Golgi?

5. An 8-cell stage embryo with GFP labeling DNA so we can watch cells divide Claudiu Giurumescu

6. Model Organisms C. elegans - genetically mapped and able to target specific cells for study Drosophila– most common model organism Lab mouse – easy to alter genetically • Easy to manipulate for experiments • Short life-cycle • Easy to keep alive • Short generation times E. coli – easy to transform

7. Our model organism: C. elegans • simple nervous system: hundreds in worms to billions in humans • old enough to reproduce in 3 days; lifespan is ~2 weeks • small - adults measure 1mm long • transparent or “see through” • hermaphrodites make genetics easy • can be grown in the lab eggs sperm reproductive, digestive, nervous, excretory

8. Adult hermaphrodite lies on its side and moves in a wave pattern

9. fusion zone my thesis topic chemical signal electrical signal A simplified synapse: how neurons communicate presynaptic neuron synaptic cleft vesicles loaded with neurotransmitter target cell

10. Puev-3-GFP GFP as a tool for visualizing which cells proteins are present in p Trujillo, et al. unpublished data.

11. How do we get DNA into the worm?By injecting worm gonads

12. muscle cells A protein in synaptic vesicles is labeled with GFP synaptobrevin-GFP

13. We can use our “marker” to find genes important for synapse formation Nakata, et al. unpublished data.

14. Team Jin/Chisholm Katsu Nakata, RIKEN Japan

15. Fluorescent Protein Activities Bacterial Transformation Protein Purification

16. Discovery of GFP-1960’s Aequorea victoria OSAMU SHIMOMURA Co-winner of Nobel Prize

17. How Fluorescence Works www.worldnetcams.com/sealife/cerianthus.jpg

18. 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.

19. Many organisms have the ability to fluoresce www.worldnetcams.com/sealife/cerianthus.jpg Jellyfish Amphipod Spider’s palps

20. Roger Tsien and Rainbow Proteins

21. E. coli

22. Bacterial chromosome What is Transformation? Uptake of foreign DNA, often a circular plasmid Plasmid

23. What is a plasmid? • A small circular piece of DNA • Naturally occurring • Can be altered in lab to express protein of interest

24. Bacterial chromosome What is Transformation? Uptake of foreign DNA, often a circular plasmid Plasmid

25. Bacterial chromosome What is Transformation? Uptake of foreign DNA, often a circular plasmid Plasmid

26. Bacterial chromosome What is Transformation? Uptake of foreign DNA, often a circular plasmid Plasmid Allow bacteria to grow for 1-3 days on plate with ampicillin. Bacterial chromosome

27. Bacterial chromosome What is Transformation? Uptake of foreign DNA, often a circular plasmid Plasmid Allow bacteria to grow for 1-3 days on plate with ampicillin. Bacterial chromosome Bacteria now express cloned fluorescent protein…

28. How are plasmids engineered? DNA Plasmid Vector Host DNA fragments (i.e. coral or jellyfish FP coding DNA) Ligate (paste) fragments into cut DNA vector Cut plasmids open with restriction enzymes + Cut genomic DNA into fragments Screen for and select plasmid containing FP gene

29. Bacterial Transformation Procedure

30. Ca++ O Ca++ O P O Base O O CH2 Sugar O Ca++ O O P Base O O CH2 Sugar OH Shielding the charge • CaCl2 • Positive charge of Ca++ ions shields negative charge of DNA phosphates

31. Stress through heat • Incubate on ice slows fluid cell membrane • Heat-shock increases permeability of membranes • Leave in heat 45 seconds!!! • Too short, and bacteria won't let in plasmid. • Too long, and the bacteria will die.

32. 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

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

34. Fluorescent Proteins-Applications

35. The rainbow of mFruit Fluorescent Proteins