Green Fluorescent Protein: A Reporter Molecule Transformation of pGLO plasmid Purification of GFP PAGE Analysis of Purified GFP (if we have time)
DNA RNA Protein Trait Central Framework of Molecular Biology • 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
Where Does It Come From? • Aquatic origin • Aequorea victoria • About 120 light emitting organs • Means of visual communication • Predation • Mating • Symbiosis • Warning signal
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.
GFP’s ChromophoreChromophores are also called fluorophores! • Composed of Ser-Gly-Tyr amino acid sequences • Oxygenating the molecule helps it to fluoresce under a ‘black light’
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 The flurophore is embedded in the beta barrel structure Absorbs light at 395 and 470 nm and emits light at 509 nm (green light) Why a Black Light? In the Organism
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
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). What’s a Reporter Molecule?
The Lab There are 3 parts to this laboratory! • Transformation of pGLO plasmid • Purification of GFP • PAGE Analysis of Purified GFP
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
Why Perform These Steps? Ca++ O • Transformation solution = CaCI2 • Positive charge of Ca++ ions shields negative charge of DNA phosphates Ca++ O P O Base Base O O CH2 Sugar Ca++ O Ca++ O O P Base O O CH2 Sugar OH
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
Luria-Bertani (LB) broth Medium that contains nutrients for bacterial growth and gene expression Carbohydrates Amino acids Nucleotides Salts Vitamins What’s LB?
Transform the pGLO plasmid into E. coli Be sure to follow the directions…exactly as they appear in the protocol. TransformationUptake of foreign DNA, often a circular plasmid GFP Beta-lactamase Ampicillin Resistance pGLO plasmids
Transcription Regulation • Lactose operon • Arabinose operon • pGLO plasmid
ara Operon lac Operon araC B A D LacI Z Y A Effector (Arabinose) Effector(Lactose) araC B A D LacI Z Y A RNA Polymerase RNA Polymerase B A D araC Z Y A Transcriptional Regulation Effector = Regulatory Molecule
ara GFP Operon ara Operon araC GFP Gene araC B A D Effector(Arabinose) Effector (Arabinose) araC B A D araC GFP Gene RNA Polymerase RNA Polymerase B A D araC araC GFP Gene Gene Regulation
Make +pGLO cultures Aerate Equilibrate HIC beads & prepare a tube of HIC resin 2. Preparation for Purification of GFP
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
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! Why Use HIC?
Hydrophobic Interaction ChromatographyThe 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
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 H H O O - - - - H H + + O O O O S S N N H H H H O O + + O O - - - - O O S S O O - - O O + + Step 1 – HIC
Wash less hydrophobic from column with low salt buffer Less hydrophobic E. coli proteins fall from column GFP remains bound to the column Hydrophobic bead H O - - + H O O S N H H O + + + + O - - - - - O S O - O + + + + Step 2 - HIC
Elute GFP from column by adding a no-salt buffer GFP Released from column matrix Flows through the column + + + + + - - - - - + + + + + Step 3 - HIC Hydrophobic bead
GFP Purification Day 3 Day 1 Day 2
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
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
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