What is next after pGLO™ bacterial transformation?

Stan Hitomi
Instructors Coordinator – Math & Science
Principal – Alamo School
San Ramon Valley Unified School District
Danville, CA

Kirk Brown
Lead Instructor, Edward Teller Education Center
Science Chair, Tracy High School
and Delta College, Tracy, CA

Bio-Rad Curriculum and Training Specialists:

Sherri Andrews, Ph.D.
sherri_andrews@bio-rad.com

Essy Levy, M.Sc.

essy_levy@bio-rad.com

Leigh Brown, M.A.

leigh_brown@bio-rad.com
Why Teach
Bacterial • Powerful teaching tool
Transformation
and Protein • Laboratory extensions
Purification?
• Real-world connections

• Link to careers and industry

• Standards based
Workshop
Time Line • Introduction

• Background on GFP

• Protein Electrophoresis of GFP

• Purify GFP using column chromatography

Starting Point:
Transformation
Procedure
Overview

Day 1

Day 2
…
Discovery of GFP

• Originally Isolated from the jellyfish

Aequorea victoria

• Naturally occurring in many

bioluminescent jelly fish, reef corals
and marine crustaceans

• Recombinant GFP has 239 amino acids

• Expressed as a 26,870 Dalton protein

• Barrel structure with the fluorescent

chromophore at center of the protein

• “Nobel prize-winning” molecule!

What is a
chromophore?
The GFP
chromophore is
comprised of
three adjacent
amino acids.
A group of atoms
and electrons
forming part of an These amino
organic molecule
that causes it to acids are
be colored enzymatically
converted to an
active cyclic
chromophore
GFP
Chromophore

Absorbs at 395 nm

Emits at 509 nm •In vivo, GFP complexes with aequorin, a calcium-

binding protein which transfers energy, to excite
GFP

•In vitro, UV light is used to excite the GFP

chromophore, absorbing light at a wavelength of
395 nm, and emitting at a longer wavelength of
509 nm visible fluorescent green light
Recombinant
GFP
GFP has been mutated

• Increased fluorescent photostability

• Improved hydrophilicity

• Increased solubility

• Improved fluorescence

• Various colors available

• Improved use as a reporter protein

Using GFP as a biological
tracer or reporter protein

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
www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm
Protein
Electrophoresis
of GFP
Procedures
Overview
Students will
learn:
Protein • Prepare an SDS-PAGE sample and understand the
components of Laemmli buffer
Electrophoresis
of GFP • Understand protein structure and mechanisms for
protein folding and unfolding and how different
conformations can be identified using
electrophoresis

• Understand how proteins are separated during gel

electrophoresis

• Understand the use of electrophoresis in the

process of transformation to protein expression

• Understand chromophores and the basis of

protein fluorescence

• Construct a standard curve and determine the

molecular weight (MW) of an unknown protein
Sample
Preparation:
SDS-
Polyacrylamide Gel
Electrophoresis
(SDS-PAGE)
“no heat” samples

Labeled tubes: • Label four screw-capped microtubes

1. White, no heat
2. Green, no heat • Add 300 µl of Sample buffer to the two “no heat’
3. White, +heat tubes
4. Green, +heat
• Using the inoculation loop, scrape a sample (20-100
colonies) from an LB/amp (white colonies) plate and
Samples should be a transfer to the “White, no heat” tube and mix
“healthy scoop” of thoroughly
colonies
• Using the inoculation loop, scrape a sample (20-100
colonies) from an LB/amp/ara (green colonies) plate
and transfer to the “Green, no heat” tube and mix
Sample • Transfer 150 µl of the “White, no heat” mixture to
Preparation the “White,+heat” tube
SDS-PAGE
• Transfer 150 µl of the “Green, no heat” mixture to
“heat” samples the “Green,+heat tube”

• Heat the “+heat” tubes to 95°C for 5 min in a

water bath. Cool to room temperature

No heating Heating
intact chromophore denatured chromophore

functional protein non-functional protein

fluorescent protein non-fluorescent protein

Heating the samples
denatures the proteins
There is an important link between the
STRUCTURE and FUNCTION of the protein
 • Tris buffer
What is in the – provides appropriate pH
CH3

Laemmli CH2
sample buffer? • SDS (sodium dodecyl sulfate) CH2
– Solubilizes and denatures proteins CH2
– Adds a negative charge to the
CH2
protein
CH2

• DTT CH2
– (1,4-Dithiothreitol) reduces disulfide CH2
bonds, to help unfold proteins and
CH2
protein complexes
CH2
• Glycerol CH2
– Increases the density of the
CH2
samples to help samples sink into
wells of the gel O

S
• Bromophenol Blue
O O

– dye to visualize samples

O -
SDS
Load and
electrophorese
samples

30min at 200V
in 1XTGS Buffer

UV illumination Coomassie Stain

How Does
SDS-PAGE
Work?
• Denatures proteins using detergent, DTT, and
heat
s-s
• Separates proteins based on size

• Negatively charged proteins move to positive

electrode
– • Smaller proteins move faster through the gel

+
Why Use • Polyacrylamide gel has a tight matrix
Polyacrylamide
Gels to • Ideal for protein separation
Separate
Proteins? • Smaller pore size than agarose

• Proteins much smaller than DNA

– Average amino acid = 110 daltons
– Average nucleotide pair = 649 daltons
– 1 kilobase of DNA = 650 kD
– 1 kilobase of DNA encodes 333 amino acids = 36 kD

• Size measured in kilodaltons (kD)

• Dalton = mass of hydrogen molecule

= 1.66 x 10-24 gram

• Average amino acid = 110 daltons

 GFP
Visualization- During Electrophoresis Post Electrophoresis
During & Post
Electrophoresis

• Fluorescent GFP can

be visualized during
electrophoresis Fluorescent
isoform

Non-

• Coomassie stained fluorescent

isoform
gels allow for
visualization of
induced GFP proteins

Prestained bands Coomassie stained

+ UV activated GFP bands
Determining the
molecular
weights of GFPs
in different
conformations
GFP
Chromatography
Kit
GFP Purification
Procedures
Overview

Day 1

Day 2 Day 3
Why Use
Chromatography?

• To purify a single
recombinant
protein of interest
from over 4,000
naturally occurring
E. coli gene
products.
Column
Chromatography

• Chromatography
used for protein
purification
– Size exclusion
– Ion exchange
– Hydrophobic
interaction
Hydrophobic
Interaction
Chromatography:
1. Add bacterial lysate to column matrix in
(HIC) high salt buffer
Steps 1–3
2. Wash less hydrophobic proteins from column in
low salt buffer

3. Elute GFP from column with

no salt buffer
Step 1:
Hydrophobic
Interaction
Chromatography
Hydrophobic
• Add bacterial lysate bead
to column matrix in
high salt buffer
– Hydrophobic
proteins interact
with column
– Salt ions interact
with the less
hydrophobic
proteins and H2O O
- O S O- H
+ - +
O N H
- O S O- H O
+ - + H H
N H +
O O
H H O

-
+
O

S
O

-
O O
-

O
- S

O
Step 2:
Hydrophobic
Interaction
Chromatography Hydrophobic
bead
• Wash less
hydrophobic from
column with low
salt buffer
– Less hydrophobic
E. coli proteins fall
from column
– GFP remains bound + -
+ -
to the column +
+
O H
- O S O- + - + H
N
+ -
O H H
+ +
O
O
-
S

O
O
-
Step 3:
Hydrophobic
Interaction
Chromatography Hydrophobic
• Elute GFP from bead
column by adding a
no-salt buffer
+
GFP -
– Released from + - +

+
column matrix

+
– Flows through the +

-
column
+ - -
+

+ +
Laboratory
Quick Guide
Helpful Hints:
Hydrophobic
Interaction • Add a small piece of
Chromatography 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
Column Both methods separate proteins from a
complex mixture
Chromatography
vs. SDS-PAGE
for protein Column Chromatography SDS PAGE
isolation and
analysis Used to isolate a protein
from a complex mixture of
An analytical technique
used to detect the
molecules based on its presence of a protein of
physical and/or chemical interest
properties
Separates molecules
HIC separates molecules based on size
based on hydrophobicity
Can compare denatured
Need to lyse open the and intact proteins to
cells to run the soluble study protein structure
proteins over the column
High concentration of
Very dilute concentration GFP from whole cell
of GFP in the HIC column lysate samples (dense
fractions colonies)

Both techniques, used in concert can help

scientists purify and analytically study proteins
Transformation
is only the pGLO Transformation
beginning…

… More
techniques are
necessary to
fully understand
the structure and Protein Purification
nature of a
protein.

Size and structure determination Results may

lead to more
experiments!
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