GREEN FLORESCENCE PROTEIN T.A: Riham Baoum

1. GREEN FLORESCENCE PROTEINT.A: Riham Baoum

2. Introduction: • GFP: a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to UV light.

3. Discovery of GFP (Osamu Shimomura) • 1960s -1970s: GFP & aequorin, were first purified from Aequorea victoria jellyfish . • GFP fluorescence occurs when aequorin protein interacts with Ca2+ ions, inducing a blue glow. Some of this luminescent energy is transferred to the GFP, shifting the overall color towards green

4. GFP Cloning (Douglas Prasher) • 1992: the cloning & nucleotide sequence of GFP in Hemoglobin Gene. • http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html#DNA

5. Expression in living organisms (Martin Chalfie) • 1994: express the coding sequence of GFP, with the first few amino acids deleted, in heterologous cells of E. coli and C. elegans.

6. Development & Improvement (Roger Tsien) • 2009: Tsien reported a new class far-red and infrared fluorescent proteins that have great potential in in vivo imaging. • Fluorescing faster than wtGFP, brighter & have different colors

7. Nobel Prize • In 2008, Osamu Shimomura, Marty Chalfie and Roger Tsien won the Nobel Prize in chemistry.

8. GFP Applications • Fluorescent protein tag. • Biological marker. • Viability assay. • Study of biological processes (example: synthesis of proteins). • Localization and regulation of gene expression • Screenable marker to identify transgenic organisms. • Green mouse? GFP cloned & expressed in almost all organisms. • Fluorescence microscopy

9. GFP transformation & purification • Transform bacteria with pGLO plasmid • pGLO™ Bacterial Transformation Kit • Purify GFP using column chromatography • (GFP) Chromatography Kit

10. What is Transformation? • change caused by genes, involves the insertion of a gene into an organism in order to change the organism’s trait. • Fundamental to the process of genetic transformation of bacteria are plasmids • What is a plasmid? • A circular piece of autonomously replicating DNA • Originally evolved by bacteria • May express antibiotic resistance gene or be modified to express proteins of interest

11. pGLO plasmid • 3 genes of interest: • GFP gene • Codes for the GFP protein • Bla gene • Codes for the enzyme B-lactamase • Ampicillin resistance • araC regulator protein • Controls expression of GFP

12. Bacterial Cell Bacterial Transformation Chromosomal DNA Plasmids

13. Methods of Transformation • Electroporation • Electrical shock makes cell membranes permeable to DNA • Calcium Chloride/Heat-Shock • Chemically-competent cells uptake DNA after heat shock

14. Transformation procedure • Treat cells with CaCl2 solution: neutralize the repulsive negative charges of the phosphate backbone of DNA & the phospholipids of the cell membrane, allowing the DNA to adhere to the cells. This is referred to ‘competent’. • 2.Heat shock: increase the permeability of the cell membrane, allowing the cells to take-in the plasmid DNA. • 3.Nutrient broth incubation in the absence of ampicillin: allows B-lactamase expression, allow cells to survive when they are subsequently placed on agar plates containing ampicillin.

15. Transformation Procedure • Step 1 Prepare appropriate plates • Step 2 Suspend cells in CaCl2 solution • Step 3 Add pGLO plasmid to cells/put onto ice • Step 4 Heat Shock at 42oC /put onto ice • Step 5 Add nutrient broth to cells • Step 6 Streak cells on to appropriate plates

16. Obtain one tube of frozen competent E. coli cells & place it on ice. • After it thaws gently resuspend the cells by flicking the bottom of the tube & Label as +pGLO. • Transfer 50 L of the cells to another tube & label it –pGLO. • place 3 L of the plasmid solution in the +pGLO tube. Close the tube and mix the DNA into the cell suspension by gently flicking the side of the tube with your finger. Return the tube to the ice. • DO NOT add plasmid DNA to the -pGLO tube.

17. Incubate the tubes on ice for 10 minutes.

18. Label 4 LB nutrient agar plates on the bottom as follows: • LB/amp plate: + pGLO • LB/amp/ara plate: + pGLO • LB/amp plate: - pGLO • LB plate: - pGLO

19. Heat shock • By using a microtube rack as a holder, transfer both the (+) pGLO and (-) pGLO tubes into the water bath, set at 42 ˚C, for exactly 50 seconds. • Place both tubes back on ice & Incubate tubes on ice for 2 minutes.

20. Place tubes on the bench top. • Add 250 μl of LB nutrient broth to the tubes & close it. • Incubate the tubes for 40 minutes at 37 ˚C.

21. Tap tubes with finger to mix! • By using a new sterile tip for each transfer, transfer 100 μl of the transformation & control suspensions onto the appropriate nutrient agar plates.

22. Spread the suspensions evenly around the surface of the lb nutrient agar. • Use a new sterile loop for each plate. do not press into the agar.

23. Stack up your plates and tape them together and place them upside down in the 37°C incubator until the next day

24. Result: • White colony: All cells grow since there is no antibiotic on the plate • No growth: Cells without plasmid don’t have antibiotic resistance. Can’t grow on media with antibiotic added • Cells with pGLO plasmid GROW & GLOW: can grow on media with antibiotic GLOW on media with arabinose (turns on GFP gene) • Lawn: Cells with plasmid have antibiotic resistance gene so can grow on media with antibiotic

25. PGLO Transformed E.coli +pGLO LB/amp +pGLO LB/amp/ara - pGLO LB/amp - pGLO LB

26. GFP Chromatography Kit • To purify a single recombinant protein of interest from over 4,000 naturally occurring E. coli gene products. • Chromatography used for protein purification • Size exclusion • Ion exchange • Hydrophobic interaction