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Green Fluorescent Protein: A Reporter Molecule
Transformation of pGLO plasmid Purification of GFP PAGE Analysis of Purified GFP (if we have time)
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Transformation and Purification of Green Fluorescent Protein (GFP)
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Central Framework of Molecular Biology
DNA RNA Protein Trait 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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Where Does It Come From? Aquatic origin Aequorea victoria
About 120 light emitting organs Means of visual communication Predation Mating Symbiosis Warning signal
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GFP Structure – The Beta Barrel
238 amino acids Cylindrical fold Very stable structure that is resistant to denaturing Alpha helices are red and beta pleated sheets are green.
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GFP’s Chromophore Chromophores are also called fluorophores!
Composed of Ser-Gly-Tyr amino acid sequences Oxygenating the molecule helps it to fluoresce under a ‘black light’
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Why a Black Light? The flurophore is embedded in the beta barrel structure Absorbs light at 395 and 470 nm and emits light at 509 nm (green light) In the Organism an influx of Ca+2 causes the first protein, aequorin, to become excited and transfer the energy to the second protein, GFP, which loses the energy by emitting a photon of green light
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GFP As A Biological Tracer
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The Nobel Prize in 2008 In 2008, Osamu Shimomura, Marty Chalfie and Roger Tsien won the Nobel Prize in chemistry for isolating GFP and using it as a ‘reporter molecule’ in biotechnology. Osamu Shimomura Martin Chalfie Roger Tsien
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What’s a Reporter Molecule?
A reporter molecule is one protein (like GFP) linked to the protein you are interested in studying. You can follow what your protein is doing by following the reporter molecule (GFP).
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The Lab There are 3 parts to this laboratory!
Transformation of pGLO plasmid Purification of GFP PAGE Analysis of Purified GFP
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General Transformation Procedure
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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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Why Perform These Steps?
Transformation solution = CaCI2 Positive charge of Ca++ ions shields negative charge of DNA phosphates Ca++ O Ca++ O P O Base Base O O CH2 Sugar Ca++ O Ca++ O P O Base O O CH2 Sugar OH
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Why Perform These Steps?
2. Incubate on ice - slows fluid cell membrane 3. Heat-shock - Increases permeability of membranes 4. Nutrient broth incubation - Allows beta-lactamase (amp resistance) expression
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What’s LB? Luria-Bertani (LB) broth
Medium that contains nutrients for bacterial growth and gene expression Carbohydrates Amino acids Nucleotides Salts Vitamins
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Transformation Uptake of foreign DNA, often a circular plasmid
GFP Transform the pGLO plasmid into E. coli Be sure to follow the directions…exactly as they appear in the protocol. Beta-lactamase Ampicillin Resistance pGLO plasmids
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Transcription Regulation
Lactose operon Arabinose operon pGLO plasmid
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Transcriptional Regulation
Effector = Regulatory Molecule RNA Polymerase Z Y A LacI Effector (Lactose) lac Operon B A D araC RNA Polymerase Effector (Arabinose) ara Operon
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Gene Regulation B A D ara Operon ara GFP Operon GFP Gene
araC RNA Polymerase Effector (Arabinose) ara Operon RNA Polymerase araC ara GFP Operon GFP Gene Effector (Arabinose)
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2. Preparation for Purification of GFP
Make +pGLO cultures Aerate Equilibrate HIC beads & prepare a tube of HIC resin
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Lecture #2
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3. Purification of GFP Purify GFP using hydrophobic interaction chromatography (HIC) Lyse GFP cells Incubate in high-salt binding buffer This turns the GFP molecule inside out to reveal hydrophobic chromophore GFP chromophore binds to HIC resin Release GFP from resin and restore structure View fluorescence
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Why Use HIC? To purify a single recombinant protein of interest from over 4,000 naturally occurring E. coli gene products. AKA…to get lots of pure product!
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Hydrophobic Interaction Chromatography The Steps
Add bacterial lysate to column matrix in high salt buffer Wash less hydrophobic proteins from column in low salt buffer Elute GFP from column with no salt buffer
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Step 1 – HIC 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 N H + - O S N H + - O S
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Step 2 - HIC Wash less hydrophobic from column with low salt buffer
E. coli proteins fall from column GFP remains bound to the column Hydrophobic bead - + - + N H + - O S - +
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Step 3 - HIC 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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GFP Purification Day 3 Day 1 Day 2
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Helpful HIC Hints 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
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4. PAGE electrophoresis
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SDS Page 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 M W G M W G M W G Fluorescent isoform Non-fluorescent isoform During Electrophoresis Post Electrophoresis Prestained bands + UV activated GFP Fluorescent bands Coomassie stained bands
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Any Questions?
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