1. Secrets of the rainforest

2. Tisha’s Story

3. GFP – Where Does It Come From?
Jellyfish: Aequorea victoria
Used for:
Means of visual communication
Predation
Mating
Symbiosis
Warning signal

4. GFP Structure – The Beta Barrel
238 amino acids
Cylindrical fold
Very stable structure that is resistant to denaturing
Alpha helices are red and beta pleated sheets are green.

5. Why a Black Light?
The flurophore is embedded in the beta barrel structure
In the Organism
An influx of Ca+2 causes the first protein, aequorin, to become excited and transfer the energy to GFP
GFP loses the energy by emitting a photon of green light

6. What’s a Reporter Molecule?
A reporter molecule is one protein (like GFP) linked to the protein you are interested in studying.
You can follow what your protein is doing by following the reporter molecule (GFP).

7. GFP As A Biological Tracer

8. Our experiment

9. Step 1 – Bacterial Culturing

10. Today – Plate culture
Used to isolate individual colonies

11. How do you streak a plate?

12. Tomorrow – broth culture
Used to grow many of one particular colony (same genetic makeup)

13. Step 2 – Lyse Bacterial Cells (to extract protein)

14. How will we lyse the cells?
Centrifuge
Lysosyme
Freezer

15. Step 3 – GFP Purification via Hydrophobic Interaction Chromatography

16. How does the HIC column work?

17. HIC Step 1 Add bacterial lysate to column matrix in high salt buffer
Hydrophobic proteins interact with column
Salt ions interact with the less hydrophobic proteins and H2O
Hydrophobic
bead N H + - O S N H + - O S

18. HIC Step 2 Wash less hydrophobic from column with low salt buffer
bead
Wash less hydrophobic from column with low salt buffer
Less hydrophobic
E. coli proteins fall from column
GFP remains bound to the column - + - + N H + - O S - +

19. HIC Step 3 Elute GFP from column by adding a no-salt buffer GFP
Hydrophobic
bead
Elute GFP from column by adding a
no-salt buffer
GFP
Released from column matrix
Flows through the column - + - + - + - + - +

20. Helpful HIC Tips
Add a small piece of paper to collection tube where column seats to insure column flow
Rest pipet tip on side of column to avoid column bed disturbance when adding solutions
Drain until the meniscus is just above the matrix for best separation