pGLO Transformation

Lab

Tak Maga
San Marin High School
STEM Marin
March 20, 2018
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PGLO TRANSFORMATION LAB

Purpose
This lab was conducted in order to gain hands-on experience with genetic engineering
techniques, practices, and ideas. In addition, basic laboratory skills were developed and
enhanced.

Hypothesis

Plate Growth Glow

+ DNA/LB ✅ 🆇

+ DNA/LB/AMP ✅ 🆇

+ DNA/LB/AMP/ARA ✅ ✅

- DNA/LB ✅ 🆇

- DNA/LB/AMP 🆇 🆇

Figure 1: Expected Results from the Transformation if 5 plates were tested, each for growth
and for glowing under an UV light

Procedure
All practices, materials, and procedures were performed in accordance with Bacterial
Transformation with Green Florescent Protein (pGLO). Bay Area Biotechnology Education
Consortium. December 2017.

How Can We Show Genetic Engineering in Cells?

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Data
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Figures 2-6: Results of the lab. Figures 2-3 are control (-DNA), figures 4-6 are + DNA. Figures
2 and 4 are LB plates. Figures 3 and 5 are LB/AMP plates. Figure 6 is a LB/AMP/ARA plate.
Figures 2 and 4 experienced growth and have visible signs of bacteria.

Observations
Not all timings and procedures were done exactly (such as to the second).
While waiting for the bacteria to be absorbed, the plates were left open for several
minutes.
While spreading the bacteria along the surface of the plate, it was not possible to
spread it in an efficient way. As a result, it is likely that there were pools of bacteria in
certain areas.
The inoculation loops were sufficiently sterilized and were cool before transferring the
DNA and bacteria into our samples.
The plates, except for stated times, were kept close to avoid contamination.
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Analysis

The data that was gathered carries several implications. The plates that were grown could
have exhibited visible growth, or, could have exhibited growth and glowed under an ultraviolet
(UV) light.

Plate Growth Glow

+ DNA/LB ✅ 🆇

+ DNA/LB/AMP 🆇 🆇

+ DNA/LB/AMP/ARA 🆇 🆇

- DNA/LB ✅ 🆇

- DNA/LB/AMP 🆇 🆇

Figure 7: Analysis of the plates

The samples were in two categories; either - DNA (without the pGLO plasmid), or + DNA
(with the pGLO plasmid). There were also three types of plates. The first was solely the DNA
and the LB. These conditions should have allowed most organisms to grow on it. The second
set of plates had an addition of ampicillin (ara), an antibiotic that would kill all bacteria that did
not have natural resistance. The third and final set of plates (which only the + DNA were
exposed to) had the sugar arabinose added to it, allowing the GFP gene in the pGLO plasmid
to be expressed. This should have resulted with glowing bacteria when viewed under the UV
light.
The positive DNA in the LB plate did experience growth. The LB nutrients in the plate
allowed the E. coli to flourish. There was no glow being emitted from the bacteria, however,
as there was no arabinose sugar present in the plate, the bacteria did not glow.
The positive DNA in the LB/AMP plate did not fare any better. Despite the pGLO’s gene
giving it resistance to the antibiotic, the bacteria did not appear to grow on the plate. Possible
errors are listed in a following section.
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The positive DNA in the LB/AMP/ARA plate did the same as the other two. Although the
conditions appeared to be prime for bacterial growth, there was none observed. The
arabinose sugar that should have then allowed the GFP gene, in the inserted plasmid, to be
expressed was not put into play.
The other exception to this pattern of no growth was found in the -DNA/ LB plate. It was
found to have visible bacterial growth. This was expected. There were no restrictions on
growth, such as ampicillin. The LB provided sufficient nutrients for the E. Coli. There was no
glow, as the DNA did not have the pGLO plasmid, and hence neither the GFP gene.
Another expected result was found in the -DNA/LB/AMP plate. There was no observed
growth of the bacteria. While there were sufficient nutrients, the presence of ampicillin killed
off any growth. Unlike the + DNA, the control samples did not have the gene that would have
allowed for antibiotic resistance.
An overall evaluation of our hypothesis results in it being incorrect. Only three of our 5
predictions were shown to be true. All + DNA plates were predicted to grow, and only one of
them matched this expectation. As a result, it is possible to conclude that there were errors
made in carrying out the lab procedure.
There are several areas in which these errors may have happened. The most likely
explanation of the lab’s failure was an incorrect transformation procedure. Based upon the
fact that no + DNA survived the plates that had ampicillin, it is probable that the DNA did not
successfully transform inside the bacteria. As a result of this, the bacteria would not have any
resistance to the ampicillin and die. Mistiming in the heat shock stage of the procedure could
have occurred, or an incorrect temperature would have produced the results that have been
observed.
However, there are also other possible explanations. The plates could have been made
incorrectly, and with too high levels of ampicillin. This would have resulted in the bacterial
death on the two plates which should have grown.
The 2 + DNA with AMP plates may have been contaminated, but again, it is unlikely that
only that set of plates could have been affected. Another issue would be not sufficiently
cooling the + DNA. This, while conceivable, is a less than realistic scenario.
The final explanation could have been materials that were not proper for use. The DNA
could have been faulty, or the ampicillin improper. This is likely this least probable
explanation, as other groups in all likeliness experienced growth that our samples were
lacking in.
Without any concrete results, it is not really possible to reflect accurately on improving the
lab. The lack of growth, probably due to the lack of initial bacteria, may have been due to the
procedure instructions. The more probable answer, however, is human error.
The results of this lab provided no conclusive results. As a result of this, a further
investigation might be to redo the lab. If possible, a larger sample would be beneficial, as it
would examine what specific item or action was at fault during our lab, or whether the results
observed were merely a product of chance.
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Conclusion
From the result of the lab, the only possible conclusion that can be drawn is that
transformation procedures are not always successful. In the experiment, 5 plates of bacteria
were tested. Of those, 3 underwent a transformation procedure for the pGLO plasmid. One of
both + and – DNA were plated on LB, and LB/AMP plates. One + DNA sample was plated on
a LB/AMP/ARA plate. From our observations, only one out of an expected 3 + DNA plates
experienced bacterial growth. Both the + DNA/AMP and + DNA/AMP/ARA plates did not
experience any growth. If the transformation procedure was carried out correctly, then the
bacteria should have had a resistance to the ampicillin that were in the other two plates.
Therefore, it can be said that the transformation procedure was not performed correctly, or
that it did not work in its desired manner. It is likely that it was not carried out correctly, due to
the amount of specific actions that needed to be done in an efficient manner. The analysis
shows many issues with the procedure that may have been done incorrectly. Among these
include mistiming (such as taking out the DNA and bacteria too soon), not sufficiently cooling
the bacteria and DNA before and after heating (not completely on ice) or having the water
bath at an incorrect temperature (too low). All of these issues conspire to make the
transformation procedure one that is not always performed correctly.