GENETIC TRANSFORMATION OF BACTERIA WITH
THE GENE FOR GREEN FLUORESCENT PROTEIN (GFP)
LAB BAC3
Adapted from "Biotechnology Explorer pGLO™ Bacterial Transformation Kit
Instruction Manual". (Catalog No. 166-0003-EDU) BIO-RAD Laboratories, 2000 Alfred
Nobel Drive, Hercules CA 94547.
STANDARDS ADDRESSED
3.1.10A/3.1.12A Apply concepts of systems and control at the molecular level to
assess the outcome of bacterial transformation.
3.2.10B/3.2.12B Describe the transformation results using precise quantitative
and qualitative skills based on observations. Evaluate the
experimental data correctly within experimental limits.
3.3.10C/3.3.12C Describe how genetic information is inherited and expressed at t
the molecular level.
KEY CONCEPTS: Genetic transformation, plasmid DNA, cloning, restriction
enzymes, antibiotic selection, gene regulation, transcription, protein expression
INTRODUCTION
Genetic transformation is the process by which a gene or genes from one organism are
transferred to another organism via an engineered molecule of DNA. If the procedure is
successful, the organism is capable of producing the protein encoded by the transformed
gene, thus creating a genetic change. Genetic transformation is commonly used in
biotechnology. In agriculture, transformation is used to confer genes for pest, frost and
spoilage resistance. Transformation of the human insulin gene into bacteria has allowed
for production of the protein on a large scale. 1 To aid in bioremediation of oil spills,
bacteria are transformed with genes that allow them to digest toxic components of the
oil. 2 The procedure contained in this lab will allow for the transformation of bacteria
with a gene from the bioluminescent jellyfish, Aequorea victoria. A successful
transformation will result in the expression of the green fluorescent protein (GFP) in the
bacteria, causing them to glow bright green under long-wave UV light.
1
2
http://www.littletree.com.au/dna.htm
http://www.epa.gov/ORD/NRMRL/lrpcd/esm/oil_spill_bioremediation_researc.htm
_______________________________________________________________________
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MICRO3-1
Transformation of Bacteria with GFP
Transformation and Antibiotic Selection: Genetic transformation in this laboratory will
be facilitated by using the pGLO plasmid (see below). A plasmid is a circular, selfreplicating DNA molecule which can be contained in a bacterial host cell without
interfering with the function of the bacterial
chromosome. Bacteria are capable, on their own, of
randomly acquiring small pieces of DNA from their
environment, but the process is inefficient. The
transformation protocol in this lab uses a chemical,
calcium chloride (CaCl2), plus heat to increase the
efficiency of DNA uptake by the bacterial cell.
Even with the chemical transformation procedure, not
every bacterial cell will incorporate the pGLO
Figure 1
plasmid into the bacteria, not every cell will receive a
copy of the plasmid. To isolate only the cells containing the pGLO DNA, the plasmid
contains the beta-lactamase gene which encodes for an ampicillin resistance (Ampr)
protein. After the transformation, the cells are grown on a solid medium called an agar
plate. This medium will contain the antibiotic ampicillin. In the presence of the
ampicillin, only the bacteria containing the pGLO plasmid will have the Ampr protein
which will break down the antibiotic, and be able to grow (Fig. 2). This process is called
antibiotic selection.
Figure 2. Transformed Bacterium: This is a diagram of what is occurring inside a bacterium
transformed with the pGLO plasmid. The bacterium is plated on agar medium containing ampicillin.
To grow, the bacterium must contain a pGLO plasmid and be expressing the ampicillin resistance
protein, β-lactamase (Ampr). The ampicillin is inactivated by the β-lactamase protein, allowing the
bacterium to grow on medium containing the antibiotic.
= pGLO plasmid
=Ampr
= transcription &
translation
Bacterial chromosome
pore
= ampicillin/
inactivation
Cloning a Gene: Plasmids can be engineered to carry a variety of genes that are not
endogenous to the host cell, like the GFP gene. A plasmid usually starts out as a very
small piece of DNA that contains a replication origin, an antibiotic resistance gene and a
cloning region, an area of the DNA that has multiple unique restriction enzyme sites.
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Transformation of Bacteria with GFP
Restriction enzymes are proteins which recognize specific DNA sequences and will
cleave, or cut, the DNA backbone at these sequences. Once the DNA backbone is
cleaved, it is possible to add, or clone, new DNA into this site. The diagram of the pGLO
plasmid (Fig. 1) shows restriction sites for the enzymes NdeI, EcoRI, and HindIII,
enzymes that are part of the original cloning site on the plasmid. The pGLO plasmid also
carries the gene araC, which produces a protein needed for transcription of genes in the
presence of arabinose sugar. The purpose of this protein is discussed below.
Transcriptional Gene Regulation: In many cases, a researcher may want to control when
a cloned gene is producing mRNA, and the corresponding protein. Proteins called
transcription factors are frequently used by cells to turn transcription “on” or “off”
depending on environmental conditions. The transcription, or production of mRNA, of
the pGLO gene is controlled by using a promoter that is only active in the presence of the
sugar arabinose (Fig. 3). The AraC protein, encoded by the araC gene on the pGLO
plasmid, is the transcription factor necessary for this control. This protein is bound at the
pGLO promoter site, but without arabinose is in the incorrect conformation, or shape, to
recruit RNA polymerase and initiate transcription. (See figure below) In the presence of
arabinose, the sugar binds to the AraC protein and changes its conformation so that in
combination with RNA polymerase, transcription is initiated and an mRNA transcript is
produced. In bacteria, transcription and translation, or protein synthesis, occurs
simultaneously, and the GFP protein is produced.
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Transformation of Bacteria with GFP
Fig. 3 Regulation of the GFP Gene
promoter
AraC
GFP
A. In a cell with no arabinose present, the AraC protein, bound to the DNA, is in
the wrong configuration to recruit RNA polymerase to the transcriptional promoter.
This means the promoter is “off” and there is no transcription of the GFP gene.
arabinose
GFP
AraC
B. A bacterium is able to take up arabinose when it is present in the environment.
Once inside the cell, arabinose binds to the AraC protein, changing its shape, or
conformation, on the DNA. This new protein conformation recruits RNA
polymerase to the transcriptional promoter.
RNA Pol
AraC
GFP
C. The binding of the RNA polymerase turns the promoter “on” and allows
transcription of the GFP gene.
mRNA transcript
RNA Pol
AraC
GFP
D. Transcription of the GFP produces an mRNA transcript. In bacteria,
transcription and translation (synthesis of proteins) can occur simultaneously, and
the GFP protein is produced within the cell.
In this lab, it is important to confirm which cells have received the plasmid, and under
which conditions the β-lactamase and GFP proteins are being produced (Fig. 4). When
the pGLO transformations are plated on agar medium containing ampicillin and
arabinose, a series of controls will be plated as well (Fig. 5). Two transformations will be
performed: one with pGLO plasmid (+pGLO) and one without the plasmid present (pGLO). A portion of the –pGLO transformation is plated on an agar medium without
ampicillin or arabinose. This control is to be sure the bacteria are viable after the
chemical and heat transformation procedure. This plate should be covered with a
bacterial “lawn”. Another portion of the –pGLO transformation is plated on an agar plate
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Figure 4. Expression of GFP: This is a diagram of what is occurring inside a bacterium transformed
with the pGLO plasmid. The bacterium is plated on agar medium containing ampicillin and arabinose.
To grow, the bacterium must contain a pGLO plasmid and be expressing the ampicillin resistance
protein, β-lactamase. In the presence of arabinose, transcription of the GFP gene is enabled, resulting in
expression of the GFP protein.
= pGLO plasmid
=Ampr
= GFP protein
Bacterial chromosome
= transcription &
translation
pore
containing ampicillin. No bacteria should grow on this plate. If it does, it means the
bacterial culture has acquired resistance and is no longer suitable for use in this
experiement. The +pGLO transformation is also plated on two different types of media.
The first portion of the transformation is plated on agar containing ampicillin only. This
control proves that the transcriptional control of the GFP gene is intact, and no GFP
protein is produced in the absence of the arabinose sugar. The final plate is the
experimental plate, containing both ampicillin and arabinose. The bacteria on this plate
are the only ones that should glow when exposed to long-wave UV light.
Figure 5: Controls for transformation of the pGLO plasmid
LB = positive control
for cell growth
LB amp= negative control
for cell growth
LB amp = transformation;
Ampr w/o GFP expression
LB amp/ara = transformation;
Ampr and GFP production
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-pGLO Plasmid
HB101 cells
+pGLO Plasmid
HB101 cells
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Transformation of Bacteria with GFP
PROCEDURES
It is important to remember that sterile technique is extremely important for this lab. All
the materials provided are sterile and proper handling of these supplies and reagents
should result in minimal contamination problems.
Lab Station Materials
2 sterile 1.5 mL microtubes
Sterile transfer pipets (10)
Sterile loops (8)
Ice water baths
LB plate (black stripe)
LB amp/ara plate (green stripe)
Sterile LB broth (600 µL)
Lab tape
Sharpie pen
Sterile CaCl2 transformation solution (600 µL)
pGLO plasmid DNA
Foam microtube rack
2 LB amp plates (red stripe)
Water bath @ 42°C
30°- 37°C Incubator (optional)
Microtube rack
Procedure
1. There are 2 sterile 1.5 mL microfuge tubes at the lab station. Label one tube
“+pGLO”, the other “-pGLO”.
2. Locate the tube labeled “Transformation Solution”; this contains a solution of CaCl2.
With a sterile pipet, aliquot 250 µL transformation solution to both the -pGLO and
+pGLO tubes. Place both tubes in an ice water bath.
3. The lab station will contain a starter plate
of HB101 bacteria. These bacteria contain
only chromosomal DNA. Take notes on how
these bacteria look in visible and UV light for
comparison with the transformed bacteria later
in the lab. The lab station will also include
pre-packaged sterile loops (look like a very
small soap bubble wand). Remove a sterile
loop from the package being careful not to
touch the loop on anything outside the bag.
Remove the cover from the starter plate and pick a single colony of the HB101 bacteria.
Immediately place the loop into the transformation solution in the tube marked “+pGLO”
and spin the loop until the entire colony is dispersed into the liquid. Check to be sure
there are no bacterial clumps floating in the transformation solution; cell clumps will
negatively affect the efficiency of the transformation. Place the tube back in the ice water
bath when the cell suspension is complete.
4. Using a new sterile loop, repeat the procedure in Step 3 for the “-pGLO” tube of
transformation solution.
5. Inspect the pGLO plasmid solution with the UV lamp provided and note your
observations. What do you expect to see at this step? Take the 2-20 µL pipettor, set it to
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Transformation of Bacteria with GFP
5 µL. Carefully remove 5 µL of the pGLO plasmid solution and add the DNA into the
“+pGLO” cell suspension. Mix the plasmid with the bacterial cells by tapping the tube
gently on the bench top. Close the tube and return it to the ice water bath. Why do you
not add plasmid DNA to the tube labeled “-pGLO”?
6. Incubate both the “+pGLO” and the “-pGLO” tubes in the ice water bath for 10 min.
Make sure the bottom of the tube is pushed through the foam rack and is in contact with
the ice water.
7. While the tubes are incubating on ice, label the 4 agar plates at your lab station as
follows:
Color code: red stripe
green stripe
red stripe
black stripe
Be sure to label the bottom of the plates (the portion containing the agar). It is very
important to label each plate correctly! Remember, the color code on the side of the agar
plates indicates which medium already contains the ampicillin for antibiotic selection and
arabinose for GFP expression. It will be confusing to analyze the results if you have
mislabeled the plates!
8. To transform the plasmid DNA into the bacteria, the cells must undergo a heat shock.
This is performed by removing the foam rack from the ice water bath and placing it
rapidly in a 42°C water bath. Incubate
the tubes at 42°C for exactly 50
seconds. Again, it is important that
the tubes are pushed down in the rack
so that the bottom of the tubes have
optimal contact with the 42°C water.
After the heat shock, immediately
place the foam rack back in the ice
water and incubate for a further 2 min.
9. Remove the foam rack from the ice water bath and place the tubes in the microtube
rack on the bench. Add 250 µL of LB broth to each tube, close the cap and gently tap the
“+pGLO” and “-pGLO” tube to mix the contents. Incubate the tubes for 10 min at
room temperature. This step of the procedure allows the cells to recover from the heat
shock treatment before performing the next part of the experiment. It also lets the cells
that have acquired a pGLO plasmid begin to express the β-lactamase protein (for
ampicillin resistance) before the cells are placed on plates that contain ampicillin.
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Transformation of Bacteria with GFP
10. Using a new sterile pipet for each tube, pipet 100 µL of the transformation and
control suspensions onto the appropriate plates
11. Spread the suspensions evenly around the agar plate by quickly sliding the flat loop
surface back and forth across the plate surface. Turning the plate in a circular motion with
your fingers while swishing the loop back and forth aids in spreading the bacterial
suspensions
evenly. Do
not press
down too
firmly or
you will
gouge the
surface of
the agar plate. This can complicate both the growth and analysis of the bacteria on the
plates. Remember to use a new sterile loop for each plate! Let the plates sit on the bench
for 2-3 minutes to allow the suspensions to soak into the plate.
12. Stack the plates and tape them together. Label the tape with the
group name and class period, if necessary. Turn the plates upside
down (with agar at top of plate) and place in a 37°C incubator
overnight. If an incubator is not available, the plates may be grown on
the bench top for 2-3 days.
Stack of upsidedown plates:
Incubate at 37°C
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DATA & ANALYSIS SHEETS
Name:________________________
Class: ________________________
Date: ________________________
Day 1: Predictions & Questions
a. Which agar plate should contain the most colonies? The least? What do these two
control plates tell us?
b. On which plate(s) should the bacterial colonies glow when examined with the UV
light?
c. One of these plates will contain bacteria that contain the pGLO plasmid, but will not
fluoresce when examined with the UV light. Which plate is this, and why won’t the
colonies glow?
d. In the “-pGLO” tube, there was no DNA present to transform into the cells. Why does
this tube need to go through the heat shock procedure?
Day 2: Data Collection and Analysis of Results
Observe the results you obtained from the transformation lab under normal room lighting.
Then turn out the lights and hold the ultraviolet light over the plates.
1. Carefully observe and draw what you see on each of the four plates in the table below.
Record your data to allow you to compare observations of the “+ pGLO” cells with your
observations for the non-transformed E. coli. Write down the following observations for
each plate.
2. How much bacterial growth do you see on each plate, relatively speaking?
3. What color are the bacteria?
4. How many bacterial colonies are on each plate (count the spots you see)?
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Data Collection Table:
General growth
# bacteria
color (vis/UV light)
Analysis of Data:
1. On Day 1, you made predictions about what would happen on each plate in terms of
bacterial growth. How does the data collected compare with your predictions? If there
are significant differences, what do you think caused them?
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Transformation of Bacteria with GFP
2. Both of the plates that contain the “-pGLO” samples are controls. What information is
given by these plates that aids in interpreting the “+pGLO” plates?
a. “-pGLO” on LB agar:
b. “-pGLO” on LB amp agar:
3. Very often an organism’s traits are caused by a combination of its genes and its
environment. Think about the green color you saw in the genetically transformed
bacteria:
a. What two factors must be present in the bacteria’s environment for you to see
the green color? (Hint: One factor is in the plate, the other is in how you observe
the bacteria.)
b. What do you think each of the two environmental factors you listed above are
doing to cause the genetically transformed bacteria to turn green?
c. What advantage would there be for an organism to be able to turn on or off
particular genes in response to certain conditions?
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Transformation of Bacteria with GFP
4. Your and your partner performed the pGLO transformation lab, correctly plated the
bacteria after transformation and placed your carefully labeled plates in the 37°C
incubator. Unfortunately, as an April Fools Day prank, another student wiped all the
identifying labels off the plates with an organic chemical. Assume the transformation
was successful. Based on your predictions and the data you collect from the plates, is
there a way to deduce which plate is which? Which plates contain bacteria with the
transformed pGLO plasmid? Which plate is the LB agar? Which plates contain
ampicillin, arabinose or both? What factors help you make your decision?
# bacteria
88
Appearance of Bacteria
room light
under UV
tan
green
“Lawn”
tan
white
No growth
--
--
tan
white
93
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pGLO? Amp/Ara?
BAC3-12
Transformation of Bacteria with GFP
EXTENSION ACTIVITY
Calculate Transformation Efficiency
Transformation efficiency is a way of calculating how many bacterial cells actually
received the pGLO plasmid during the transformation procedure. In many experiments,
like gene therapy, optimizing the number of cells receiving new genetic material is very
important. There are two important pieces of information you will need to determine the
transformation efficiency.
1. The total number of green fluorescent colonies growing on the LB amp/ara plate.
This is data you have already obtained when analyzing your plates.
_____ = # of green fluorescent colonies
2. The total amount of pGLO plasmid DNA in the bacterial cells spread on the LB
amp/ara plate. This part of the calculation has a several steps which will determine the
amount of pGLO plasmid spread on the LB amp/ara plate.
a. The stock solution of pGLO plasmid DNA is:
80 ng
μL
or 0.08 μg
μL
b. The total amount of pGLO plasmid added to the transformation tube is
calculated by multiplying:
(concentration of DNA in µg/µL) x (volume of DNA in µL) = DNA in µg
Or
5 µL X 0.08
μg
= ______µg = Total pGLO in transformation
μL
c. Since not all the DNA you added to the bacterial cells will be transferred to the
agar plate, you need to find out what fraction of the DNA was actually spread
onto the LB amp/ara plate. To do this, divide the volume of the transformation
solution spread on the LB amp/ara plate by the total volume of liquid in the test
tube containing the DNA.
Calculate the total transformation solution volume:
CaCl2 Solution =
pGLO plasmid solution =
LB broth
=
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µL
Volume on plate = ____ µL
µL
µL
µL total volume
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Fraction of transformation = Volume spread on LB amp/ara plate (µL) = ______
used
Total transformation volume in tube (µL)
d. The fraction of transformation solution gives a percentage. To calculate the
amount of pGLO plasmid on the plate, multiply this percentage (from c.) by the
total µg pGLO DNA (from b.).
(Fraction of transformation) X (total µg pGLO) = pGLO plasmid on plate (µg)
____________ X __________µg = _____ µg
e. The final calculation is to determine the number of colony forming units (cfu)
per ug DNA transformed. Divide the number of colonies counted on the LB
amp/ara plate (see Step 1) by the µg DNA determined in 2d. (above).
# colonies = _____________ = __________ cfu
µg DNA
µg
µg
The transformation efficiency will vary greatly depending on the method of
transformation used. It is almost always expressed in scientific notation. For
example, if you calculate a transformation efficiency of 550 cfu/µg, then you
would write this as:
5.5 x 102 cfu/µg
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Flow Chart of pGLO Transformation Protocol
= Overnight step
Label two tubes
“+pGLO” and
“-pGLO”. Add 250
µL transformation
solution to each. Place
tube in ice water bath
_
+
_
+
Inoculate each tube with HB101 bacterial colony
from starter plate. Disperse bacteria evenly and
place tubes back in ice water bath
_
+
Add 5 µL of the pGLO plasmid to the
“+pGLO” tube. Mix well and place
tube back in ice water bath.
-pGLO
LB
Incubate tubes on
ice for 10 min.
-pGLO
LB amp
Label LB agar plates
during incubation.
+pGLO
LB amp
+pGLO
LB amp/ara
+
_
Incubate tubes on
ice for 2 more min.
Heat shock transformation mix
tubes in 42°C water bath for 50 sec.
+
_
Remove tubes from ice & place on bench.
Add 250 µL LB broth to each. Incubate
for 10 min at room temperature.
(Go to next page.)
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Use a sterile spreader to plate
100 µL of appropriate
transformation mix on each
labeled agar plate.
_
LB = positive
control for growth
LB amp= negative
control for growth
+
LB amp = transformation;
Ampr w/o GFP expression
LB amp/ara =
transformation; Ampr and
GFP production
Grow plates overnight at 37°C. Do
not overgrow or individual colonies
may be difficult to count.
LB = positive
control for growth
LB amp= negative
control for growth
LB amp = transformation;
Ampr w/o GFP expression
LB amp/ara =
transformation; Ampr and
GFP production
Analyze results of plates in the following class period.
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