By: Jathuya Peranantharuban and Leeza Yackballick
What’s Glowing?
1.
Recall what you observed when you shined the UV
light source onto a sample of
original pGLO plasmid DNA and describe your
observations.
When the UV light was shined on the sample
of the original pGLO plasmid DNA, no fluorescence
colour was observed, and did not change in appearance under the UV light.
2.
Which of the two possible sources of the
fluorescence can now be eliminated?
The original bacteria and the pGLO plasmids
DNA can be eliminated since no fluorescence colour was observed under the UV
light.
3.
What does this observation indicate about the
source of the fluorescence?
This observation indicates that the source
of of fluorescence is related to the protein in the DNA of the transformed
plasmids. The fluorescence green light was only observed in the DNA of the
bacteria that contains the pGLO plasmid DNA, LB nutrient, Ampicillin and
Arabinose. This observation indicates
the source of fluorescence is from the gene that codes for green fluorescent
protein from the bioluminescent jellyfish Aequorea victoria. This gene allows
the transformed bacteria to glow a bright green colour under ultraviolet light.
4.
Describe the evidence that indicates whether
your attempt at performing a genetic transformation was successful or not
successful.
The evidence that would indicate whether our
attempt at performing a genetic transformation was successful or not depends on
the end observations. If the transformed bacteria display the desired
characteristic then we have successfully performed genetic transformation. In
this case, if the plate with +pGLO LB/amp/ara
and +pGLO LB/amp have colonies of bacteria, and the colonies on the +pGLO
LB/amp/ara plate should fluoresce bright green under UV light. If the
experiment was unsuccessful then colonies of bacteria will not be present on
the +pGLO LB/amp/ara and +pGLO LB/amp
plates. Errors that may affect the
results include not transferring appropriate number of colonies to the plates
or mistakes in following the instructions as indicated in the procedure.
The Interaction between Genes and Environment
Some E.coli did grow on the LB plate that
does not contain ampicillin or
arabinose.
1.
From your results, can you tell if these
bacteria are ampicillin resistant by looking at them on the LB plate? Explain
your answer.
From our results, we cannot tell if these
bacteria are ampillicin restistent by looking at them on the LB plate. We are not able to distinguish the colonies
that are ampicillin resistant and those that are ampicillin sensitive since
they look similar. The bacteria on the LB plate and the bacteria on the LB/amp
plate look alike, so therefore there is not a physical feature to indicate
whether they are ampicillin resistant.
2.
How would you change the bacteria’s environment
to best tell if they are ampicillin resistant?
To best tell if they are ampicillin
resistant, we can change the bacteria’s environment. To test whether the
bacteria are resistant to ampicillin, some bacteria can be transferred from the LB plate to the
LB/amp plate. If the bacteria are able to grow on the LB/amp plate, this would
show the bacteria are resistant to ampicillin, whereas if the bacteria do not
survive, they are ampicillin sensitive. The plasmids that are able to survive contain
the gene resistant to the antibiotic, and therefore when the plasmids are
placed in the new environment with the antibiotic, the colonies can continue to
grow.
3.
Very often an organism’s traits are caused by a
combination of its genes and the environment it lives in. 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 and the other factor is in how you look
at the bacteria).
The two factors that must be present is the
appropriate solutions and environment. The sugar arabinose must be present to
activate the GFP gene. This gene is next to the arabinose promoter, so it
becomes active in the presence of arabinose. In addition, UV light is
necessary to cause the GFP protein to fluoresce.
b. What do you think each of the two
environmental factors you listed above are doing to cause the genetically
transformed bacteria turn green?
When these two factors are present, the
transformed bacteria display the gene from the jellyfish. The original
plasmid contained three different genes ,ara A, ara B, and ara D that code for
three enzymes needed to convert arabinose to a form that can be used. A fourth
gene, araC, codes for a protein that acts to control the structural genes. When
arabinose is present, it initiates transcription by promoting the
binding of RNA polymerase. The arabinose interacts with araC and causes araC to
change its shape. In the transformed plasmids, the promoter (PBAD)
and the araC gene are present but the ara A, ara
B, and ara D genes are replaced by the GFP gene. Therefore, in the
presence of arabinose, araC protein promotes the binding of RNA polymerase and GFP
is produced. Furthermore, the long-wave UV light give the gene the energy to
fluoresce the green colour.
c. What
advantage would there be for an organism to be able to turn on or off particular
genes in response to certain conditions?
Gene regulation is an important feature
that allows organisms to survive and prevent producing unnecessary proteins.
This allows the organism to produce proteins only when needed and according the
their environment. Moreover, control of the gene
is important to allow a cell to create the gene products it needs when it needs
them. This gives cells the flexibility to adjust to a new environment, external
signals, damage to the cell, food, etc. Examples include the Lac operon in E.Coli,
a negative control system, that breaks down lactose when present. Another example is a positive
control system since E.coli need tryphospho to produce proteins, the Trp
operon is usually “on”.
