Green Fluorescent Protein: A Reporter Molecule

3.

1.

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 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

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 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’

Why a Black Light?

 The flurophore is embedded in the beta barrel structure

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  Absorbs light at 395 and 470 nm and emits light at 509 nm ( 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?

1.

Transformation solution = CaCI 2  Positive charge of Ca++ ions shields negative charge of DNA phosphates

Ca ++ Ca ++ O O P O O CH 2 O Sugar Base Ca ++ Ca ++ O O P O CH 2 O O Sugar Base 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.

pGLO plasmids

Beta-lactamase

Ampicillin Resistance

Transcription Regulation

 Lactose operon  Arabinose operon  pGLO plasmid

Transcriptional Regulation

Effector = Regulatory Molecule

LacI LacI lac Operon Z Y A Effector (Lactose) Z Y A Z RNA Polymerase Y A ara C ara Operon B A D Effector (Arabinose) araC araC B A D RNA Polymerase B A D

Gene Regulation

ara C ara Operon B A D Effector (Arabinose) araC araC B A D RNA Polymerase B A D araC ara GFP Operon GFP Gene Effector (Arabinose) araC araC GFP Gene RNA Polymerase 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 H 2 O

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 O O 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

Day 1

GFP Purification

Day 2 Day 3

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

LB/amp

 Bacterial lysates are prepared in Laemmli buffer  Samples are loaded onto polyacrylamide gels

LB/amp/ara

GFP Visualization-During & Post Electrophoresis  Samples are electrophoresed

M W G M W G M W G

 Fluorescent GFP can be visualized during electrophoresis  Coomassie stained gels allow for visualization of induced GFP proteins Fluorescent isoform Non-fluorescent isoform

During Electrophoresis Prestained bands + UV activated GFP Fluorescent bands Post Electrophoresis Coomassie stained bands

Any Questions?