pGLO™ Transformation and Purification of Green Fluorescent Protein (GFP)

Discovery of GFP Naturally produced in Jellyfish– Aequorea victoria Discovered in 1960’s

Osamu Shimomura Martin Chalfie Roger Y. Tsien Osamu Shimomura first isolated GFP from the jellyfish Aequorea victoria, Martin Chalfie demonstrated the value of GFP as a luminous genetic tag for various biological phenomena. Roger Y. Tsien contributed to our general understanding of how GFP fluoresces. He also extended the colour palette beyond green allowing researchers to give various proteins and cells different colours. Osamu Shimomura Martin Chalfie Roger Y. Tsien

Osamu Shimomura: first isolated GFP from the jellyfish Aequorea victoria,

Martin Chalfie: C. elegans glowing with green fluorescent protein

Roger Tsien: range of fluorescent proteins have brought some colour to laboratory work.

The barrel structure of GFP The tertiary structure of GFP is barrel-like, consisting of 11 beta sheets depicted as the green ribbons and an internal chromophore of three adjacent amino acids, depicted as green spheres

Cyclization of the tripeptide Ser-Tyr-Gly: The active chromophore of GFP is comprised of three adjacent amino acids in the primary amino acid chain. The three amino acids are enzymatically converted to an active cyclic chromophore in vivo

Excitation and emission profiles of the GFP chromophore

Using GFP as a biological tracer http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html With permission from Marc Zimmer

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

Microtubule dynamics in pombe

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

Bacterial Transformation Cell wall GFP Bacterial chromosomal DNA Beta lactamase (ampicillin resistance) pGLO plasmids

Gene Expression Beta Lactamase Ampicillin resistance Green Fluorescent Protein (GFP) Aequorea victoria jellyfish gene araC regulator protein Regulates GFP transcription

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

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)

Now, prepare 3 kinds of plates LB 1. LB 2. LB + Amp+ (3 plates) 3. LB + Amp+ + Arabinose (2 plates) LB/Amp LB/Amp/Ara

1: LB plates 2、3、4: LB +Amp+plates 5、6、7: LB+ Amp++Arabinose plates

Ampicillin will be degraded in high temperature! Store concentration Working concentration Amp+ 10 mg/ml 50mg/ml Arabinose 200mg/ml 50mg/ml LB/Amp 50ml LB medium+250ml Amp+ 50ml LB medium+250ml Amp++12.5ml Arabinose LB/Amp/Ara Ampicillin will be degraded in high temperature!

Central Framework of Molecular Biology DNA RNA Protein Trait

What is Transformation? GFP Uptake of foreign DNA, often a circular plasmid Beta-lactamase Ampicillin Resistance

Methods of Transformation Electroporation Electrical shock makes cell membranes permeable to DNA Calcium Chloride/Heat-Shock Chemically-competent cells uptake DNA after heat shock

Reasons for Performing Each Transformation Step? Ca++ O Ca++ O P O Base O O CH2 Sugar Transformation solution = CaCI2 Positive charge of Ca++ ions shields negative charge of DNA phosphates O Ca++ O P O Base O O CH2 Sugar OH

Why Perform Each Transformation Step? Cell wall GFP 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)

Transformation Procedure Prepare 2 tubes, add 250ml of 50mMCacl2 Suspend 2-4 bacterial colonies in Cacl2 add 1ml of 0.2mg/ml pGLO plasmid DNA to one tube, the other one leave as control Extra competent cells: Add 1ml of pGLO plasmid Place tubes on ice（10mins） Heat-shock at 42°C for 50sec~1min and place on ice（1min） Add 250ml LB medium and Incubate at 37oC for10 mins Centrifuge ,get rid of 300-350ml supernatant, re-suspend, Streak plates Incubate LB plates in incubator o/n

Transformation Procedure Overview Day 1 Day 2

Make sure to streak to correct plates! extra competent cells

Grow? Glow? Follow protocol On which plates will colonies grow? LB/Amp Follow protocol On which plates will colonies grow? Which colonies will glow? LB/Amp/Ara LB