* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Green Fluorescent Protein: A Reporter Molecule
Deoxyribozyme wikipedia , lookup
Photosynthetic reaction centre wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Real-time polymerase chain reaction wikipedia , lookup
Epitranscriptome wikipedia , lookup
Biosynthesis wikipedia , lookup
Endogenous retrovirus wikipedia , lookup
Community fingerprinting wikipedia , lookup
Interactome wikipedia , lookup
Metalloprotein wikipedia , lookup
Gene therapy of the human retina wikipedia , lookup
Magnesium transporter wikipedia , lookup
Chromatography wikipedia , lookup
Point mutation wikipedia , lookup
Gel electrophoresis wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Transcriptional regulation wikipedia , lookup
Transformation (genetics) wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Gene regulatory network wikipedia , lookup
Western blot wikipedia , lookup
Nuclear magnetic resonance spectroscopy of proteins wikipedia , lookup
Protein structure prediction wikipedia , lookup
Size-exclusion chromatography wikipedia , lookup
Biochemistry wikipedia , lookup
Proteolysis wikipedia , lookup
Bimolecular fluorescence complementation wikipedia , lookup
Protein–protein interaction wikipedia , lookup
Expression vector wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Gene expression wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Green Fluorescent Protein: A Reporter Molecule 1. 3. Transformation of pGLO plasmid 2. Purification of GFP PAGE Analysis of Purified GFP (if we have time) Transformation and Purification of Green Fluorescent Protein (GFP) Central Framework of Molecular Biology DNA RNA Protein 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 Trait 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 Alpha helices are red and beta pleated sheets are green. 238 amino acids Cylindrical fold Very stable structure that is resistant to denaturing GFP’s Chromophore Chromophores are also called fluorophores! Composed of SerGly-Tyr amino acid sequences Oxygenating the molecule helps it to fluoresce under a ‘black light’ 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 GFP As A Biological Tracer 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 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). The Lab There are 3 parts to this laboratory! Transformation of pGLO plasmid Purification of GFP PAGE Analysis of Purified GFP General Transformation Procedure 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? Transformation solution = CaCI2 1. Ca++ O ++ O P O CaBase O CH2 Base O Sugar Positive charge of Ca++ ions shields negative charge of DNA phosphates Ca++ Ca++ O O P O Base O CH2 O 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 What’s LB? Luria-Bertani (LB) broth Medium that contains nutrients for bacterial growth and gene expression Carbohydrates Amino acids Nucleotides Salts Vitamins 1. 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 pGLO plasmids Resistance Transcription Regulation Lactose operon Arabinose operon pGLO plasmid Transcriptional Regulation Effector = Regulatory Molecule ara Operon lac Operon LacI Z Y A ara C Z Y A araC Y A B A D RNA Polymerase RNA Polymerase Z A D Effector (Arabinose) Effector (Lactose) LacI B araC B A D Gene Regulation ara GFP Operon ara Operon ara C B A D araC Effector (Arabinose) Effector (Arabinose) araC B A D araC RNA Polymerase araC B A D GFP Gene GFP Gene RNA Polymerase araC GFP Gene 2. Preparation for Purification of GFP 1. 2. 3. Make +pGLO cultures Aerate Equilibrate HIC beads & prepare a tube of HIC resin Lecture #2 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 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! Hydrophobic Interaction Chromatography The Steps 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 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 Step 2 - HIC Wash less hydrophobic from column with low salt buffer Less hydrophobic E. coli proteins fall from column GFP remains bound to the column O - O S OO Hydrophobic bead Step 3 - HIC Elute GFP from column by adding a no-salt buffer GFP Released from column matrix Flows through the column 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 4. PAGE electrophoresis 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 MWG MWG M W G Fluorescent GFP can be visualized during electrophoresis Fluorescent isoform Coomassie stained gels allow for visualization of induced GFP proteins Non-fluorescent isoform During Electrophoresis Prestained bands + UV activated GFP Fluorescent bands Post Electrophoresis Coomassie stained bands Any Questions?