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pGLO™ Transformation and Purification of Green Fluorescent Protein (GFP)
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Discovery of GFP
Naturally produced in Jellyfish– Aequorea victoria
Discovered in 1960’s
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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.
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Osamu Shimomura: first isolated GFP from the jellyfish Aequorea victoria,
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Martin Chalfie: C. elegans glowing with green fluorescent protein
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Roger Tsien: range of fluorescent proteins have brought some colour to laboratory work.
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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
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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
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Excitation and emission profiles of the GFP chromophore
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Using GFP as a biological tracer
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.htmlWith permission from Marc Zimmer
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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
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Microtubule dynamics in pombe
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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
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Bacterial Transformation
Beta lactamase(ampicillin resistance)
pGLO plasmids
Bacterial chromosomal DNA
Cell wall
GFP
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GeneExpression
• Beta Lactamase– Ampicillin resistance
• Green Fluorescent Protein (GFP)– Aequorea victoria
jellyfish gene
• araC regulator protein– Regulates GFP
transcription
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Transcriptional Regulation
B A DaraC
B A DaraC
RNA Polymerase
Effector (Arabinose)
araC B A D
ara Operon
RNA Polymerase
Z Y A
Z Y ALacI
Effector (Lactose)
Z Y ALacI
lac Operon
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Gene Regulation
RNA Polymerase
araC
ara GFP Operon
GFP Gene
araC GFP Gene
araC GFP Gene
Effector (Arabinose)
B A DaraC
B A DaraC
RNA Polymerase
Effector (Arabinose)
araC B A D
ara Operon
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Now, prepare 3 kinds of plates
1. LB
2. LB + Amp+ (3 plates)
3. LB + Amp+ + Arabinose (2 plates)
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1: LB plates
2、 3、 4: LB +Amp+plates
5、 6、 7: LB+ Amp++Arabinose plates
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Store concentration Working concentration
Amp+ 10 mg/ml 50g/ml
Arabinose 200mg/ml 50g/ml
50ml LB medium+250l Amp+
50ml LB medium+250l Amp++12.5l Arabinose
Ampicillin will be degraded in high temperature!
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Central Framework of Molecular Biology
DNA RNA Protein Trait
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What is Transformation?
• Uptake of foreign DNA, often a circular plasmid
GFP
Beta-lactamase
Ampicillin
Resistance
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Methods of Transformation
• Electroporation– Electrical shock makes cell membranes
permeable to DNA
• Calcium Chloride/Heat-Shock– Chemically-competent cells uptake DNA after
heat shock
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Reasons for Performing Each Transformation Step?
1. Transformation solution = CaCI2
Positive charge of Ca++ ions shields negative charge of DNA phosphates
Ca++
Ca++
OCH2
O
P O
O
OBase
CH2
O
P
O
O
O
Base
OH
Sugar
Sugar
OCa++
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Why Perform Each Transformation Step?
2. Incubate on iceslows fluid cell membrane
3. Heat-shockIncreases permeability of membranes
4. Nutrient broth incubationAllows beta-lactamase expression
Beta-lactamase(ampicillin resistance)
Cell wall
GFP
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Transformation Procedure •Prepare 2 tubes, add 250l of 50mMCacl2
•Suspend 2-4 bacterial colonies in Cacl2
•add 1l of 0.2g/l pGLO plasmid DNA to one tube, the other one leave as control
•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 300-350l supernatant, re-suspend,
•Streak plates
•Incubate LB plates in incubator o/n
Extra competent cells:
Add 1l of pGLO plasmid
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Transformation Procedure Overview
Day 1
Day 2
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extra competent cells
Make sure to streak to correct plates!
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Grow? Glow?
• Follow protocol
• On which plates will colonies grow?
• Which colonies will glow?