1. pGLO™ Transformation and Purification of
Green Fluorescent Protein (GFP)

2. Discovery of GFP Naturally produced in Jellyfish– Aequorea victoria
Discovered in 1960’s

3. Osamu Shimomura Martin Chalfie Roger Y. Tsien
Osamu Shimomura first isolated GFP from the jellyfish Aequorea victoria,
Martin Chalfie demonstrated the value of GFP as a luminous genetic tag for various biological phenomena.
Roger Y. Tsien contributed to our general understanding of how GFP fluoresces. He also extended the colour palette beyond green allowing researchers to give various proteins and cells different colours.
Osamu Shimomura Martin Chalfie Roger Y. Tsien

4. Osamu Shimomura: first isolated GFP from the jellyfish Aequorea victoria,

5. Martin Chalfie:
C. elegans glowing with green fluorescent protein

6. Roger Tsien:
range of fluorescent proteins have brought some colour to laboratory work.

7. The barrel structure of GFP
The tertiary structure of GFP is barrel-like, consisting of 11 beta sheets depicted as the green ribbons and an internal chromophore of three adjacent amino acids, depicted as green spheres

8. Cyclization of the tripeptide Ser-Tyr-Gly: The active chromophore of GFP is comprised of three adjacent amino acids in the primary amino acid chain. The three amino acids are enzymatically converted to an active cyclic chromophore in vivo

9. Excitation and emission profiles of the GFP chromophore

10. Using GFP as a biological tracer
With permission from Marc Zimmer

11. Links to Real-world GFP is a visual marker
Study of biological processes (example: synthesis of proteins)
Localization and regulation of gene expression
Cell movement
Cell fate during development
Formation of different organs
Screenable marker to identify transgenic organisms

12. Microtubule dynamics in pombe

15. 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

16. Bacterial Transformation
Cell wall
GFP
Bacterial
chromosomal DNA
Beta lactamase
(ampicillin resistance)
pGLO plasmids

17. Gene Expression Beta Lactamase Ampicillin resistance
Green Fluorescent Protein (GFP)
Aequorea victoria jellyfish gene
araC regulator protein
Regulates GFP transcription

18. Transcriptional Regulation
RNA Polymerase Z Y A
LacI
Effector (Lactose)
lac Operon B A D
araC
RNA Polymerase
Effector (Arabinose)
ara Operon

19. Gene Regulation B A D ara Operon ara GFP Operon GFP Gene
araC
RNA Polymerase
Effector (Arabinose)
ara Operon
RNA Polymerase
araC
ara GFP Operon
GFP Gene
Effector (Arabinose)

20. Now, prepare 3 kinds of platesLB
1. LB
2. LB + Amp+ (3 plates)
3. LB + Amp+ + Arabinose (2 plates)
LB/Amp
LB/Amp/Ara

21. 1: LB plates
2、3、4: LB +Amp+plates
5、6、7: LB+ Amp++Arabinose plates

22. Ampicillin will be degraded in high temperature!
Store concentration
Working concentration
Amp+
10 mg/ml
50mg/ml
Arabinose
200mg/ml
50mg/ml
LB/Amp
50ml LB medium+250ml Amp+
50ml LB medium+250ml Amp++12.5ml Arabinose
LB/Amp/Ara
Ampicillin will be degraded in high temperature!

23. Central Framework of Molecular Biology
DNA
RNA
Protein
Trait

25. What is Transformation?
GFP
Uptake of foreign DNA, often a circular plasmid
Beta-lactamase
Ampicillin
Resistance

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

27. Reasons for Performing Each Transformation Step?
Ca++ O
Ca++ O P O
Base O O
CH2
Sugar
Transformation solution = CaCI2
Positive charge of Ca++ ions shields negative
charge of DNA phosphates O
Ca++ O P O
Base O O
CH2
Sugar OH

28. Why Perform Each Transformation Step?
Cell wall
GFP
2. Incubate on ice
slows fluid cell membrane
3. Heat-shock
Increases permeability of membranes
4. Nutrient broth incubation
Allows beta-lactamase
expression
Beta-lactamase
(ampicillin resistance)

29. Transformation Procedure
Prepare 2 tubes, add 250ml of 50mMCacl2
Suspend 2-4 bacterial colonies in Cacl2
add 1ml of 0.2mg/ml pGLO plasmid DNA to one tube, the other one leave as control
Extra competent cells:
Add 1ml of pGLO plasmid
Place tubes on ice（10mins）
Heat-shock at 42°C for 50sec~1min and place on ice（1min）
Add 250ml LB medium and Incubate at 37oC for10 mins
Centrifuge ,get rid of ml supernatant, re-suspend,
Streak plates
Incubate LB plates in incubator o/n

30. Transformation Procedure Overview
Day 1
Day 2

31. Make sure to streak to correct plates!
extra competent cells

32. Grow? Glow? Follow protocol On which plates will colonies grow?
LB/Amp
Follow protocol
On which plates will colonies grow?
Which colonies will glow?
LB/Amp/Ara LB