pGLO™ Transformation and Purification of Green Fluorescent Protein (GFP)
pGLO™ Transformation and Purification of Green Fluorescent Protein (GFP)
description
Transcript of pGLO™ Transformation and Purification of Green Fluorescent Protein (GFP)
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pGLO™ Transformation and Purification of
Green Fluorescent Protein (GFP)
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DNA RNA Protein Trait
Central Framework of Molecular Biology
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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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Using GFP as a biological tracer
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.htmlWith permission from Marc Zimmer
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Transformation: Procedure Overview
Day 1
Day 2
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What is Transformation?
• Uptake of foreign DNA, often a circular plasmid
GFP
Beta-lactamase
Ampic illin
Resistance
GFP
Beta-lactamase
Ampic illin
Resistance
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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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Plasmid DNA
Bacterial cell
Genomic DNA
Bacterial DNA
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Scanning electron micrograph of supercoiled plasmid
Graphic representation
The Many Faces of Plasmids
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Gene Expression
• Beta Lactamase– Ampicillin resistance
• Green Fluorescent Protein (GFP)– Aequorea victoria
jellyfish gene
• araC regulator protein– Regulates GFP
transcription
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Bacterial Transformation
Beta lactamase(ampicillin resistance)
pGLO plasmids
Bacterial chromosomal DNA
Cell wall
GFP
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Transcriptional Regulation
• Lactose operon
• Arabinose operon
• pGLO plasmid
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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
Transcriptional Regulation
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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
Gene Regulation
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• Electroporation– Electrical shock makes cell membranes
permeable to DNA
• Calcium Chloride/Heat-Shock– Chemically-competent cells uptake DNA after
heat shock
Methods of Transformation
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Transformation Procedure
• Suspend bacterial colonies in Transformation solution
• Add pGLO plasmid DNA
• Place tubes on ice
• Heat-shock at 42°C and place on ice
• Incubate with nutrient broth
• Streak plates
http://faculty.clintoncc.suny.edu/faculty/michael.gregory/files/
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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 ice
slows fluid cell membrane
3. Heat-shock
Increases permeability of membranes
4. Nutrient broth incubation
Allows beta-lactamase
expression
Beta-lactamase(ampicillin resistance)
Cell wall
GFP
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What is Nutrient Broth?
• Luria-Bertani (LB) broth
• Medium that contains nutrients for bacterial growth and gene expression– Carbohydrates– Amino acids– Nucleotides– Salts– Vitamins
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Grow? Glow?
• Follow protocol
• On which plates will colonies grow?
• Which colonies will glow?
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Results
http://faculty.clintoncc.suny.edu/faculty/michael.gregory/files/
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Volume Measurement
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When transferring bacteria to or from an agar plate
• Organisms are transferred by using a sterile loop and reaching in from the side while keeping the plate covered as much as possible.
• This technique minimizes the risk of contamination from above.
http://faculty.clintoncc.suny.edu/faculty/michael.gregory/files/
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When adding DNA to the transformation reaction
• Immerse a sterile loop into the bottle containing plasmid DNA.
• When the center of the loop is coated with a soap-like film, transfer it the + DNA microtube
http://faculty.clintoncc.suny.edu/faculty/michael.gregory/files/