1. BACTERIAL TRANSFORMATION TRAINING
What do we see here? Living organisms (bacteria) expressing a trait (fluorescence) on a petri dish. How did someone create this picture? They used “normal” bacteria (can’t see with naked eye) and insrted genetic instructions for the production of fluorescent proteins. While this is fun, pretty, etc., there are serious scientific endeavors that utilize not only the powerful technique of transformation, but the application of fluorescent proteins. Through this protocol, students will become genetic engineers--

2. Gloriana Gallegos Trujillo
An Elegant Way to Study Developmental Neurobiology (with a little help from GFP)
Gloriana Gallegos Trujillo
Jin Lab
use gfp as a way to light up a specific cell in a living worm

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

4. Why do we use Green 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 Easy to manipulate for experiments Short life-cycle
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
eggs
sperm
reproductive, digestive, nervous, excretory
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

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

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

10. GFP as a tool for visualizing which cells proteins are present in
Puev-3-GFP
use gfp as a way to light up a specific cell in a living worm p
Trujillo, et al. unpublished data.

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

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

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
There are lots and lots of organisms found in nature that can fluoresce all on their own. Here are some examples:
Amphipods
Coral
Jellyfish
Some animals, like this spider in the lower right hand corner, even use fluorescence to attract their mates.

18. Bioluminescence vs. Fluorescence
Scorpion- UV Light
Scorpion- Natural 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
There are lots and lots of organisms found in nature that can fluoresce all on their own. Here are some examples:
Amphipods
Coral
Jellyfish
Some animals, like this spider in the lower right hand corner, even use fluorescence to attract their mates.
Jellyfish
Amphipod
Spider’s palps

20. Roger Tsien and Rainbow Proteins
Why are we here? What is a protein? What is purification?

21. E. coli

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

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

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

27. What is Transformation?
Bacterial chromosome
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. Shielding the charge CaCl2
Positive charge of Ca++ ions shields negative charge of DNA phosphates
Ca++ O
CH2 P
Base OH
Sugar

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

34. Fluorescent Proteins-Applications
Fluorescent proteins are revolutionary in bio-technology research. Tsien lab mutated FPs into a “rainbow” of colors.

35. The rainbow of mFruit Fluorescent Proteins