pglo™ transformation and purification of green fluorescent protein (gfp) · 2020-03-06 ·...
TRANSCRIPT
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pGLO™ Transformation and Purification of
Green Fluorescent Protein (GFP)
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Stan Hitomi Coordinator – Math & Science San Ramon Valley Unified School District Danville, CA Kirk Brown Lead Instructor, Edward Teller Education Center Science Chair, Tracy High School and Delta College, Tracy, CA Sherri Andrews, Ph.D.
Curriculum and Training Specialist Bio-Rad Laboratories Essy Levy, M.Sc.
Curriculum and Training Specialist Bio-Rad Laboratories
Instructors
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Why Teach
Bacterial
Transformation
and Protein
Purification?
• Powerful teaching tool
• Laboratory extensions
• Real-world connections
• Link to careers and industry
• Standards based
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pGLO™
Bacterial
Transformation
Kit
Bio-Rad pGLO Kit Advantages
• Standards-based
• Comprehensive curricula for inquiry-based
investigations
• Compatible with 50 minute class periods
• Serves entire class of 32 students
(up to 4 students per group)
• Cost-effective
• Success in student’s hands
• Safe
• Striking results!
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Green
Fluorescent
Protein (GFP)
Chromatography
Kit
GFP Purification Kit Advantages
• Cloning in action
• Links to biomanufacturing
• Biopharmaceutical development
• Amazing visual results
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Workshop
Time Line
• Introduction
• Transform bacteria with pGLO plasmid
• Purify GFP using column chromatography
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Central
Framework of
Molecular
Biology
DNA RNA Protein Trait
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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.html
With permission from Marc Zimmer
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pGLO Bacterial
Transformation
Kit
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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
Ampicillin
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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Bacterial DNA
Plasmid DNA
Bacterial cell
Genomic DNA
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The Many
Faces of
Plasmids
Scanning electron micrograph of
supercoiled plasmid
Graphic representation
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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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Transcriptional
Regulation
B A D araC
B A D ara
C
RNA Polymerase
Effector (Arabinose)
araC B A D
ara Operon
RNA Polymerase
Z Y A
Z Y A LacI
Effector (Lactose)
Z Y A LacI
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 D araC
B A D ara
C
RNA Polymerase
Effector (Arabinose)
araC B A D
ara Operon
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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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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
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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++
O CH
2
O
P O
O
O Base
CH2
O
P
O
O
O
Base
OH
Sugar
Sugar
O Ca++
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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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Laboratory
Quick Guide
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GFP
Electrophoresis
Extension
• SDS PAGE sample
preps are made
from white and
green colonies
• Bacterial lysates
are prepared in
Laemmli buffer
• Samples are loaded
onto polyacrylamide
gels
LB/amp LB/amp/ara
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GFP
Visualization-
During & Post
Electrophoresis
• Samples are
electrophoresed
• Fluorescent GFP can
be visualized during
electrophoresis
• Coomassie stained
gels allow for
visualization of
induced GFP proteins
During Electrophoresis Post Electrophoresis
M W G M W G M W G
Fluorescent
isoform
Non-fluorescent
isoform
Prestained bands
+ UV activated GFP
Fluorescent
bands
Coomassie stained
bands
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Volume
Measurement
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GFP
Chromatography
Kit
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GFP Purification
Procedures
Overview
Day 3 Day 2
Day 1
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Why Use
Chromatography?
• To purify a single
recombinant protein
of interest from
over 4,000 naturally
occurring E. coli
gene products.
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Column
Chromatography
• Chromatography
used for protein
purification
–Size exclusion – Ion exchange –Hydrophobic
interaction
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Hydrophobic
Interaction
Chromatography:
(HIC)
Steps 1–3
1. Add bacterial lysate to column matrix in high salt buffer
2. Wash less hydrophobic proteins from column in low salt buffer
3. Elute GFP from column with no salt buffer
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Step 1:
Hydrophobic
Interaction
Chromatography
• Add bacterial lysate
to column matrix in
high salt buffer
–Hydrophobic proteins interact with column
–Salt ions interact with the less hydrophobic proteins and H2O
Hydrophobic
bead
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Step 2:
Hydrophobic
Interaction
Chromatography
• Wash less
hydrophobic from
column with low
salt buffer
– Less hydrophobic E. coli proteins fall
from column –GFP remains bound
to the column
O
S
O
O O - -
Hydrophobic
bead
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Step 3:
Hydrophobic
Interaction
Chromatography
• Elute GFP from
column by adding a
no-salt buffer
GFP
–Released from column matrix
– Flows through the column
Hydrophobic
bead
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Laboratory
Quick Guide
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Helpful Hints:
Hydrophobic
Interaction
Chromatography • Add a small piece of
paper to collection tube where column seats to insure column flow
• Rest pipet tip on side of column to avoid column bed disturbance when adding solutions
• Drain until the meniscus is just above the matrix for best separation