pGLO Bacterial Transformation Laboratory – Creating Bioluminescent Escherichia coli
by: ABADILLA, Marlo Gabriel O.
Background
Bacteria can change and copy foreign DNA.
This is considered as an evolutionary
advantage as it helps them survive in their
environment ("Bacterial transformation",
2007). Bacterial DNA contain plasmids,
smaller clusters of DNA. These plasmids can
sometimes be exchanged between bacteria in
what is known as “conjugation”.
Meanwhile, genetic engineering is the
process where DNA is directly manipulated
in order to gain a certain characteristic
(“What is genetic engineering?”, 2017).
In this experiment, students do something
similar to such, as they attempt to try and
integrate the genes of the jellyfish Aequorea
victoria into E. coli.
Purpose
The study aims to apply concepts and lessons
learned in the classroom involving DNA, the
central dogma, adaptation, and more. The
experiment, despite being in a set, gave the
groups an opportunity to figure out the
experiment on their own. This was due to the
instructor giving this chance to the students.
Through the accomplishment of the
experiment, students will gain further and
more practical knowledge about how cells
absorb DNA, and how they change with it.
Variables
In this study, the independent variables were
the presence of the substances: an antibiotic
(ampicillin), a genetic modifier (pGLO), and
an activator for the plasmid (arabinose).
As for the controlled variables, there is the
use of identical E. coli HB101 K-12 for all
plates, the use of identical amounts of Luria-
Bertani (LB) broth across all sample dishes,
and the identical procedures all plates will
undergo.
Hypothesis
Different settings of each plate will produce
different results in each one.
A plate with only LB will not glow, as well
as a plate with only LB and/or ampicillin.
Next, for the manipulated plate, a plate with
LB, ampicillin, and arabinose will glow, as
they contain the pGLO plasmid, and are
activated by the arabinose.
Procedure
The procedure in this experiment follow
closely that of Bio-Rad Technology
Explorer’s “pGLO™ Bacterial
Transformation Kit: Shine a Little Light on
Your Molecular Biology — Literally!” (n.d.).
First of all, the nutrient agar was prepared
using the packs, and heating them and stirring
them on hot plates. These were then poured
into the individual petri dishes so as to give
them time to cool down.
Next, once the agar has already cooled down,
the bacteria is rehydrated, and then streaked
onto the individual plates, all with different
configurations.
The plates are first incubated at 37C
overnight. After which, the cells are
collected, and then will be used for
inoculating transformation and negative
control tubes. The transformation tube
contains the +pGLO plasmid.
The tubes are then incubated for 10 minutes
on ice. They are heat shocked at 42C for 50
seconds, then placed back in the ice for 2
minutes. Nutrient broth is added, and the
tubes are incubated at room temperature for
10 minutes. After which, the suspensions are
spread onto the plates.
The suspension with the +pGLO plasmid, the
transformation tube, is spread onto: A)
LB/AMP plate and B) LB/AMP/ARAB
plates and the suspension with no +pGLO
plasmid, the control tube, is spread onto: A)
LB/AMP plate and B) LB plates. This in
which AMP means ampicillin and ARAB
means arabinose,
After this, the plates are incubated again
overnight, and then are observed under a UV
light.
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 Photo Gallery

Figure 1.1 & 1.2. Nutrient Agar Figure 4.1 & 4.2. Ice bath and heat shock
Preparation. of vials.

Figure 5.1. Streaking of E. coli.

Figure 2.1 & 2.2. Labelling agar plates.

Figure 6.1. Observation of plates under UV

light.

Figure 3.1. Rehydration of substances.

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Data and Observations E. Interpretation
A. LB Plate All plates showed almost identical results as
predicted by the Bio-Rad Kit Manual.
The bacteria on the Luria-Bertani (LB) Plate
External factors must have affected the
grew as normal. Regular colonies were found
plates, as Set D, the LB/Amp/Arab + pGLO
on the plate after the incubation period. This
plates did not glow as prominently as
was expected as the plate was not modified in
expected.
any way by any substance except the agar.
Conclusion
B. LB|Amp
When the bacteria is exposed to the plasmid
On the plate with ampicillin, no bacteria
pGLO, the antibiotic ampicillin so that it can
grew, and like the past plate, this was also to
adopt it, and the activator arabinose, the
be expected. Ampicillin, being an antibiotic
bacterial colony will then glow. However,
for the bacteria, most likely killed .2all of it
some problems were encountered in the
on the plate before the bacteria could grow.
execution of this experiment, and that may
C. LB|Amp + pGLO have affected its results.

This plate does not contain the activator for Analysis

the pGLO plasmid. Although the pGLO was
not activated in the colonies, it is apparent
that they have absorbed foreign genetic
material, as the colonies may not have
glowed, they exhibited a whitish color that
was not apparent in the LB plate. This was
also to be expected.
D. LB|Amp|Arab + pGLO
In this plate, there was a bioluminescent
glow, however, it was fainter than expected.
It glowed due to the fact that the plasmid
together with the activator was already in the
plate. A stronger glow was expected.
In the execution of the procedure for the
experiment, more caution could have been
practiced. The incubation time was
unintentionally left unchecked by the group
of researchers, thus extending the supposed
24 hours into 48 hours.
There was also a lack of equipment to
properly perform this experiment. An
incubator was not present at the time, and so
the actual temperature that the petri dishes
were kept at is uncertain, and at the same
time, they could have been exposed to
foreign bacteria.

References

Bacterial transformation. (2007, November 16). Retrieved January 30, 2018, from
https://www.sciencelearn.org.nz/resources/2032-bacterial-transformation

Bio-Rad Biotechnology Explorer. (n.d.). Retrieved from http://www.bio-

rad.com/LifeScience/pdf/Bulletin_9563.pdf

What is genetic engineering? (2017, February 17). Retrieved from

https://www.yourgenome.org/facts/what-is-genetic-engineering