pGLO Transformation Lab

I. Introduction:
Genetic transformation is a change cause by genes, involving the insertion of a gene into an
organism to change the organism’s trait. Transformation in bacterial cells occurs when the cell
incorporates DNA into its genetic material; in a laboratory setting, this is encouraged by placing
the mixtures of transformation solution and plasmid DNA (in +pGLO tube only) on ice, then
rapidly transferring them to a hot water bath for about fifty seconds, and then placing them back
on ice again – this procedure is called heat shock. The agent which the new genetic material
is incorporated into is the bacterial plasmid. A plasmid is a circular deoxyribonucleic acid (DNA)
molecule that replicates independently of the bacterial chromosome and often is the avenue for
which a bacteria gains resistance to an antibiotic. The pGLO plasmid has been modified by
removing the structural genes of the arabinose operon (ara A, ara B, and ara D). These genes
were replaced by a gene from a bioluminescent jellyfish that codes for the production of green
fluorescent protein (GFP). This protein glows when it is illuminated by UV light. Because this
gene is adjacent to the arabinose promoter, it becomes active in the presence of arabinose. The
pGLO which will be incorporated into the genome of the E. Coli bacteria used in the lab,
provides resistance to the antibiotic ampicillin. The antibiotic ampicillin is used in this lab to
demonstrate the effect of recombinant plasmids and transformation. By applying ampicillin to a
Petri dish with and without pGLO, we can see the success of an antibiotic against bacteria as
well as the success of recombinant bacteria against an antibiotic.

II. Hypothesis
The bacteria with the +pGLO plasmids that are resistant to the antibiotic ampicillin and have the
gene for GFP will survive and grow on the transformation plates that have LB/amp. In addition,
+pGLO bacteria on a plate on the LB/amp/ara will grow and glow green under UV light because
the inclusion of arabinose. In the control plates, -pGLO bacteria that are amp sensitive will not
be able to grow on the LB/amp plates because the amp will kill the bacteria as it doesn’t have
any resistance which the recombinant plasmid gave to the other bacteria. The other control
plate with the pGLO bacteria and no amp added will host a lawn of bacteria colonies but there is
no Glowing factor so it won’t glow under UV light.

III. Procedure
1. Label one closed micro test tube +pGLO and another -pGLO. Label both tubes with your
group’s name. Place them in the foam tube rack.

2. Open the tubes and using a sterile transfer pipet, transfer 250 µl of transformation solution
(CaCl2). CaCl2 punches holes in the cell membrane, allowing small DNA molecules like
plasmids to slip through into the interior.

3. Place the tubes on ice.

4. Use a sterile loop to pick up a single colony of bacteria from your starter plate. Pick up the
+pGLO tube and immerse the loop into the transformation solution at the bottom of the tube.
Spin the loop between your index finger and thumb until the entire colony is dispersed in the
transformation solution (with no floating chunks). Place the tube back in the tube rack in the ice.
Using a new sterile loop, repeat for the -pGLO tube.

5. Examine the pGLO plasmid DNA solution with the UV lamp. Note your observations.
Immerse a new sterile loop into the plasmid DNA stock tube. Withdraw a loopful. There should
be a film of plasmid solution across the ring. This is similar to seeing a soapy film across a ring
for blowing soap bubbles. Mix the loopful into the cell suspension of the +pGLO tube. Close the
tube and return it to the rack on ice. Also close the –pGLO tube. Do not add plasmid DNA to the
-pGLO tube.

6. Incubate the tubes on ice for 10 minutes. Make sure to push the tubes all the way down in the
rack so the bottom of the tubes sticks out and make contact with the ice.

7. While the tubes are sitting on ice, label your four agar plates on the bottom (not the lid) as
follows: Label one LB/amp plate: +pGLO; Label the LB/amp/ara plate: +pGLO; Label the other
LB/amp plate: -pGLO; Label the LB plate: -pGLO.

8. Heat shock. Heat shocking renders the cell membranes even more permeable, allowing the
plasmid to penetrate the bacteria as thoroughly as possible. Using the foam rack as a holder,
transfer both the (+) pGLO and (-) pGLO tubes into the water bath, set at 42 °C, for exactly 50
seconds. Make sure to push the tubes all the way down in the rack so the bottom of the tubes
sticks out and make contact with the warm water. When the 50 seconds are done, place both
tubes back on ice. For the best transformation results, the change from the ice (0°C) to 42°C
and then back to the ice must be rapid. Incubate tubes on ice for 2 minutes.

9. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube
and, using a new sterile pipet, add 250 µl of LB nutrient broth to the tube and reclose it. LB
revitalizes the bacteria after the CaCl2 and heat shocking with its influx of nutrients. Repeat with
a new sterile pipet for the other tube. Incubate the tubes for 10 minutes at room temperature.

10. Tap the closed tubes with your finger to mix. Using a new sterile pipet for each tube, pipet
100 µl of the transformation and control suspensions onto the appropriate plates.

11. Use a new sterile loop for each plate. Spread the suspensions evenly around the surface of
the agar by quickly skating the flat surface of a new sterile loop back and forth across the plate
surface.

12. Stack up your plates and tape them together. Put your group name and class period on the
bottom of the stack and place the stack upside down in the 37°C incubator until the next day.
IV. Results

Plate Growth Glow

+pGLO – LB/amp Yes No

+pGLO – LB/amp/ara Yes Yes

-pGLO – LB/amp No No

-pGLO – LB Yes No

V. Analysis
 Total number of fluorescent cells: 640
 Total amount of pGLO plasmid DNA used: 0.16 μg DNA
 Transformation Efficiency= (Total # of transformants/ Amt DNA spread on the
agar plate) (640)/ 0.16 μg DNA = 4000 transformants/ μg

Sources of error: If correct lab technique wasn’t used, unwanted microbes would be in the
plates when they were open for too long. Then the data won’t reliable. Also, if the incubation
time wasn’t exact, that’s also room for error. Also, incorrect amount of time in the heat shock
process will result in an error as too much time may kill the bacteria.

VI. Conclusion
This experiment was successful in determining the transformation efficiency and results of
genetic transformation. The plasmid must have given resistance to the antibiotic on the plates
that contained ampicillin. The –pGLO bacteria that didn’t have the plasmid couldn’t survive on
the ampicillin plates, which resulted in no growth of the control. Another plate of –pGLO thrived
in a plate that didn’t contain the antibiotic. The two plates that contained the plasmids both had
bacterial resistance to the antibiotic. In addition, the plate that had LB/ampicillin/arabinose gave
the colonies a fluorescent green glow under ultraviolet light. Transformation efficiency is the
quantitative value that describes how effective the transfer of plasmids into bacteria. The
number represents the number of transformed colonies produced per microgram of DNA added.
My group had transformation efficiency within the range. I believe the experiment can be
improved by using a better mechanism in counting the number of fluorescent cells which will
result in a more accurate transformation efficiency result. The transformation process can be
used in many applications. For example, produce human proteins inexpensively, modify
proteins and change activity, and produce non-protein products by cloning enzymes. Examples
of gene cloning are insulin, Lung surfactant protein, and enzymes used in laundry detergent.