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: 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.htmlWith 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

Beta lactamase(ampicillin resistance)

pGLO plasmids

Bacterial chromosomal DNA

Cell wall

GFP

GeneExpression

• Beta Lactamase– Ampicillin resistance

• Green Fluorescent Protein (GFP)– Aequorea victoria

jellyfish gene

• araC regulator protein– Regulates GFP

transcription

Transcriptional Regulation

B A DaraC

B A DaraC

RNA Polymerase

Effector (Arabinose)

araC B A D

ara Operon

RNA Polymerase

Z Y A

Z Y ALacI

Effector (Lactose)

Z Y ALacI

lac Operon

Gene Regulation

RNA Polymerase

araC

ara GFP Operon

GFP Gene

araC GFP Gene

araC GFP Gene

Effector (Arabinose)

B A DaraC

B A DaraC

RNA Polymerase

Effector (Arabinose)

araC B A D

ara Operon

Now, prepare 3 kinds of plates

1. LB

2. LB + Amp+ (3 plates)

3. LB + Amp+ + Arabinose (2 plates)

1: LB plates

2、 3、 4: LB +Amp+plates

5、 6、 7: LB+ Amp++Arabinose plates

Store concentration Working concentration

Amp+ 10 mg/ml 50g/ml

Arabinose 200mg/ml 50g/ml

50ml LB medium+250l Amp+

50ml LB medium+250l Amp++12.5l Arabinose

Ampicillin will be degraded in high temperature!

Central Framework of Molecular Biology

DNA RNA Protein Trait

What is Transformation?

• Uptake of foreign DNA, often a circular plasmid

GFP

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?

1. Transformation solution = CaCI2

Positive charge of Ca++ ions shields negative charge of DNA phosphates

Ca++

Ca++

OCH2

O

P O

O

OBase

CH2

O

P

O

O

O

Base

OH

Sugar

Sugar

OCa++

Why Perform Each Transformation Step?

2. Incubate on iceslows fluid cell membrane

3. Heat-shockIncreases permeability of membranes

4. Nutrient broth incubationAllows beta-lactamase expression

Beta-lactamase(ampicillin resistance)

Cell wall

GFP

Transformation Procedure •Prepare 2 tubes, add 250l of 50mMCacl2

•Suspend 2-4 bacterial colonies in Cacl2

•add 1l of 0.2g/l pGLO plasmid DNA to one tube, the other one leave as control

•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 300-350l supernatant, re-suspend,

•Streak plates

•Incubate LB plates in incubator o/n

Extra competent cells:

Add 1l of pGLO plasmid

Transformation Procedure Overview

Day 1

Day 2

extra competent cells

Make sure to streak to correct plates!

Grow? Glow?

• Follow protocol

• On which plates will colonies grow?

• Which colonies will glow?