Nicholas Mack

3 February 2016

pGLO TRANSFORMATION LAB REPORT

Introduction: The purpose of this lab was to observe the effects of the pGLO plasmid on
various colonies of E. coli bacteria. pGLO is a genetically modified plasmid that
primarily contains three genes (with the origin of replication). The first is an ampicillin
(antibiotic) resistance gene that allows the genetically modified bacteria to grow in this
antibiotic. The second is the GFP gene that codes for the green fluorescent protein,
which, when in the right conditions, causes bacteria to emulate green light. The third
gene, araC, acts as a trigger to activate the GFP gene when the bacteria are in an
environment with this type of sugar. In this experiment, students transferred the modified
plasmid to the E. coli via transformation: the process by which a plasmid is removed
from its host and placed into a foreign bacteria cell. Once the plasmid is inside the new
cell, the bacteria are able to recognize the genes encoded in the plasmid and express a
desired phenotype. For this lab specifically, student transferred the modified plasmid to
the E. coli by using a transformation solution containing CaCl. A process called heat
shock was then used to insert the plasmid into the E. Coli. Four different growth plates
were used for bacteria growth to simulate different groswing conditions. The first plate (pGLO / LB) did not have ampicillin and contained bacteria that did not have the plasmid.
There should be a lot of growth on this plate because the bacteria will be able to feed off
of the broth (LB). However, the colonies should not glow because, in this case, a
repressor protein will be bound next to the promoter (starting point) of the GFP gene as
an acting operon, causing transcription of that gene to stop until ARA is present. This is
an example of how the GFP gene is regulated. The second plate (-pGLO / LB / AMP)
should not exhibit any growth because, since the bacteria do not have the pGLO plasmid,
they are not resistant to ampicillin, causing no growth. The third plate (+pGLO/ LB /
AMP) should exhibit moderate growth because the bacteria do contain the plasmid,
allowing them to grow on ampicillin and feed off of LB. However, the color of these cells
should remain white because the repressor will still be bounded to the operator due to the
absence of the sugar, causing transcription of the GDP gene from its promoter to be

turned off. The fourth plate (+pGLO / LB / AMP / ARA) should exhibit bacteria that are
glowing because, with ARA present, an ARA inducer molecule will be able to bind to the
repressor to inactivate it, allowing transcription of the GFP to commence. With this
experiment, students will have a better understanding of how plasmids and gene
regulation work.
Results:
Expected:

+
Source: https://sites.google.com/a/pvlearners.net/aishwarya-karlapudi/blog-1

**Note: DNA + and DNA are equivalent to saying pGLO + and pGLO -.

Actual:
Observations of E.Coli Plates With and Without pGLO
Plate Name
- pGLO / LB

Observations
1. There is a lot of growth. A lawn has
formed. Bacteria colonies are clustered.

-pGLO / LB / AMP

2. Color is white with and w / out UV.

1. As expected, there is no growth at all.
2. None with and w / out UV light

+ pGLO / LB / AMP

(obviously).
1. Moderate growth, though the
colonies are not as large as
pGLO / LB. More scattered
throughout plate.
2. Color is white with and w /out UV
1. There is a lot of growth. Bacteria

+ pGLO / LB / AMP / ARA

colonies are clustered.

2. Color of the bacteria appears to be
white without UV light and green
and glowing with UV light
*Pictures of All Plates On Separate Document
Conclusion:
After concluding this experiment, the group knew that the transformation was
successful because the E. coli glowed in the plate with + pGLO / LB / AMP / ARA,
indicating that the bacteria successfully received the plasmid with the GFP and AMP
genes. Because the environment contained the arabinose sugar, an ARA inducer molecule
was able to inactivate the repressor and allow transcription of the inserted GTP molecule
to commence. Other evidence includes the fact that the bacteria in the + pGLO / LB /

AMP plate successfully grew because they had the antibiotic resistance gene. The -pGLO
/ LB / AMP plate indicates that without this antibiotic resistance gene, the bacteria would
not be able to survive in an environment with ampicillin. In terms of improvement, what
the group could do better next time is to incubate the test tubes containing LB nutrient
broth for the specified amount of time. During the experiment, the group did not incubate
these tubes for enough time, which could have affected their results. The group will be
sure to improve their timing for the next experiment.
In this experiment, the group successfully produced genetically modified bacteria
that exhibited desired phenotypes. Each E. coli cell with the inserted plasmid was able to
regulate the expression of the GDP by utilizing an inducer molecule. Based on the results
from this experiment, there is an incredible advantage to turning on and off genes. If a
certain chemical or substance is present in an organisms environment that the organisms
cells need to digest, then a particular gene coding for digestion can be turned by
inactivating its repressor. When these enzymes are not needed, the repressor can be
simply activated, turning off transcription. Thus, the overall advantage of turning on and
off genes is that it conserves energy. By conserving energy, a cell can prioritize its duties
and become more efficient. There are numerous applications of transforming bacteria.
One potential application is that transforming bacteria can be used in bioremediation to
eliminate pollution in an environment, such as during an oil spill. Using genetic
engineering, a plasmid that contains genes for pollutant digestion can be inserted into a
specific type of bacteria. The genetically modified bacteria can be then introduced to a
specific environment where it will be able to eliminate toxins and pollutants. In the
scenario of an oil spill, genetically modified bacteria can be used to break down
hydrocarbons found in oil.

Source: http://katiebobatey0.tripod.com/bacterialtransformation/id1.html