Download Transformation and Protein Purification

TM
pGLO Bacterial Transformation
Courtesy BioRad Corporation
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pGLO/ Green Fluorescent Protein Isolation
A single bacteria might be worth 5 billion dollars to
the right buyer.
Central Framework of
Molecular Biology
DNA
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RNA Protein Trait
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What is transformation?

Uptake of foreign DNA, often a circular
plasmid, from the surroundings with the
expression of these genes. GFP
Amp Resistance
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What is a plasmid?
A
circular piece of autonomously
replicating DNA
ori
bla
Originally evolved by bacteria
 May express antibiotic resistance gene or
be modified to express proteins of interest

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Uses of Genetic
Transformation

Agriculture


Bioremediation


Gene therapy
Production of human proteins

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Introduction of genes into bacteria giving
them the ability to degrade hydrocarbons,
“Oil Eating Bacteria”
Medicine


Introduction of drought, pest, or frost
resistance genes “BT Corn”
Insulin, clotting factors
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pGLO PlasmidDraw this!!
Beta Lactamase
• Ampicillin resistance
Green
Fluorescent
Protein
• Aequorea victoria jellyfish
gene
araC
regulator protein
• Regulates
GFP transcription
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araC
ori
pGLO
bla
GFP
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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.

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Work of the
pGLO
Plasmid
Arabinose Operon
RNA Polymerase
Regulatory
Gene--araC
Binds RNA polymerase
Gene products
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RNA Polymerase
Transcriptional Regulation
ara GFP Operon
ara Operon
araC
B
A
D
araC
Effector
(Arabinose)
Effector
(Arabinose)
araC
B
A
D
araC
RNA Polymerase
araC
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B
A
D
GFP Gene
GFP Gene
RNA Polymerase
araC
GFP Gene
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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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Transformation
Procedures
Day 1
Day 2
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Reasons for performing each
transformation step?
Ca++
O
Transformation
solution = CaCl2
Ca+2
Positive charge of
ions shields negative
charge of DNA
phosphates
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Ca++
O P O
Base
O
CH2
O
Sugar
O
Ca++
O P O
Base
O
CH2
O
Sugar
OH
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Why perform each transformation
step?
Incubation on ice slows
fluid cell membranes
Cell wall
GFP
Heat-shock increases
permeability of cell membrane
Incubation in LB
nutrient broth allows beta
lactamase expression
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Beta lactamase
(ampicillin resistance)
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Inducible, Repressible or
Both??
http://faculty.clintoncc.suny.edu/faculty/Mic
hael.Gregory/files/bio%20101/Bio%20101
%20Lectures/Gene%20Regulation/gene.ht
m
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The Gel Analysis
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Graphing Migration Distance
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