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04/19/231
pGLOpGLOTM Bacterial Transformation Bacterial TransformationCourtesy BioRad CorporationCourtesy BioRad Corporation
pGLO/ Green Fluorescent Protein Isolation
A single bacteria might be worth 5 billion dollars to the right buyer.
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Central Framework of Molecular Biology
DNA RNA Protein Trait
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Uptake of foreign DNA, often a circular plasmid, from the surroundings with the expression of these genes.
What is transformation?
GFP
Amp Resistance
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A circular piece of autonomously replicating DNA
What is a plasmid?
Originally evolved by bacteria May express antibiotic resistance gene or
be modified to express proteins of interest
ori
bla
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Uses of Genetic Transformation Agriculture
Introduction of drought, pest, or frost resistance genes “BT Corn”
Bioremediation Introduction of genes into bacteria giving them
the ability to degrade hydrocarbons, “Oil Eating Bacteria”
Medicine Gene therapy
Production of human proteins Insulin, clotting factors
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pGLOori
blaGFP
araC
pGLO Plasmid-Draw this!!
Beta Lactamase• Ampicillin resistance
Green Fluorescent Protein• Aequorea victoria jellyfish
gene
araC regulator protein• Regulates GFP transcription
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Purpose of pGLO Transformation
To transform E. coli K-12 with a plasmid that contains a gene that codes for Green Fluorescent Pigment (GFP)
Transformed cells will produce GFP and glow a brilliant green under UV light.
Work of the pGLO Plasmid
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Arabinose Operon
Gene products
RNA Polymerase
RNA Polymerase
RegulatoryGene--araC
Binds RNA polymerase
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Transcriptional Regulation
araC
RNA Polymerase
Effector (Arabinose)
B
B
B
A
A
A
D
D
D
araC
araC
ara Operon
RNA Polymerase
araC
ara GFP Operon
GFP Gene
araC GFP Gene
araC GFP Gene
Effector (Arabinose)
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Transformation Procedure
Suspend bacterial colonies in Transformation Solution
Add pGLO plasmid DNA Place tubes on ice Heat shock at 42oC and place on ice Incubate with nutrient broth Streak plates
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TransformationProcedures
Day 2
Day 1
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Reasons for performing each transformation step?
Transformation solution = CaCl2
Positive charge of Ca+2 ions shields negative charge of DNA phosphates
Ca++
Ca++
OCH2
O
P O
O
O Base
CH2
O
P
O
O
O
Base
OH
Sugar
Sugar
OCa++
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Why perform each transformation step?
Incubation on ice slows fluid cell membranes
Heat-shock increases permeability of cell membrane
Incubation in LB nutrient broth allows beta lactamase expression
GFP
Cell wall
Beta lactamase(ampicillin resistance)
Inducible, Repressible or Both??
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http://faculty.clintoncc.suny.edu/faculty/Michael.Gregory/files/bio%20101/Bio%20101%20Lectures/Gene%20Regulation/gene.htm
The Gel Analysis
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Graphing Migration Distance
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