Unformatted text preview: Bacterial Transformation Abstract:
The objective of this experiment is to conduct bacterial transformation on E. coli bacteria, by
successfully inserting a plasmid, pGLO, and getting the bacteria to express a gene that allows it
to glow under UV light. This gene is called the GFP gene. The GFP gene not only allows the
bacteria to grow, but it also gives the bacteria a resistance to ampicillin. If this experiment were
to have been conducted with no errors whatsoever, the majority of the bacterial colonies should
have been able to grow and glow. Unfortunately, in this experiment that was not the case. While
none of the colonies glowed, some were still able to grow, due to the expression of the bla gene.
Although the hypothesis was not supported, it was most likely a flaw in human error and not in
the experiment itself.
Introduction:
Genes are codes inside of DNA that dictate which proteins are made within an organism. These
tiny chunks of DNA are essentially instructions that make up living organisms. Similar to
evolution, transformation occurs within nature when a certain trait becomes favorable and useful
for an organism. Genetic transformation occurs when a gene is inserted into an organism in order
to change a certain trait of that organism for the organism’s own benefit. In this experiment, a
plasmid, pGLO, will be inserted into the genome of E. coli bacteria by the process of heat shock.
This will be done so that the GFP gene will be produced, giving the bacteria the ability to glow
under UV light as well as resistance to an antibiotic—ampicillin. The production of GFP also
depends on two things: the presence of arabinose sugar and the araC gene. Not all of the petri
dishes will include the pGLO plasmid. In those that do, the bla gene will produce the bla protein.
First, the bla gene will be transcribed by the bla mRNA and then it will be translated into the bla protein. The bla protein’s purpose is to give the E. coli bacteria resistance to ampicillin. The
presence of the bla gene will enable growth in the bacteria in some cultures, but not in others.
The other cultures that the E. coli bacteria will not be able to grow in will be those lacking the
presence of the bla gene. The same thing that happened to the bla gene will also happen to the
araC gene. The araC gene will first be transcribed by the araC mRNA and then translate into the
araC protein. The araC protein is able to either positively or negatively regulate the expression of
proteins and, in this case, the protein that is being regulated for this experiment is the GFP
protein. The relationship between the araC protein and the GFP protein is directly related. In the
presence of the araC protein the GFP protein is expressed, while in the absence of the GFP
protein it is not expressed. In theory, without any experimental errors, the samples expressing
both the GFP gene and the araC protein will express the bla protein, giving the bacteria
resistance to ampicillin, as well as allow it to glow under UV light. The samples that do not
express the bla protein will die in the presence of ampicillin, hence no growth or glow will occur.
Materials and Methods:
First, two micro test tubes will be needed. One of the test tubes will be used for those petri dishes
with the pGLO included in it and the other one will be for those petri dishes without the pGLO in
it. Make sure to label these so that the test tubes do not get mixed up—separation of the pGLO
samples is necessary for this experiment. Place the two micro test tubes into a foam tube rack
and use a pipette to fill each of them with 250 uL (microliters) of transformation solution and
close both of the lids on each of the micro test tubes. Then, take the foam rack with the tubes in it
and place it in a covered ice bucket for three minutes. After three minutes, remove the foam rack
from the ice and open a new, sterile loop from its package and remove it from the opposite end of where the loop is located. Use the loop to pick up a single colony of E. coli, open one of the
micro test tubes’ lid and submerge the loop into the transformation solution. Using a new, sterile
loop for each trial, repeat this step for both of the micro test tubes, the -pGLO and the +pGLO.
After both of the micro test tubes have been contaminated with the E. coli place them back into
the covered ice bucket for another three minutes. This time, after removing the foam tray from
the ice bucket, only open the the +pGLO tube—leave the -pGLO tube closed. Grab another new,
sterile loop and insert it into the pGLO plasmid DNA stock tube. Stir the loop in the solution and
when it is removed there should be a film over the loop, which indicates that the DNA has been
picked up by the loop. Stick the loop into the +pGLO tube ONLY and stir. Remove the loop and
close the lid on the +pGLO once again, and place the foam tray with the tubes back into the ice.
This time, make sure that the tubes are pushed all the way down in the foam rack and that the
tubes are fully submerged in the ice. The tubes will sit in the ice for ten minutes. While waiting
for the tubes to be done in the ice bucket, acquire the following plates: 1 LB plate (no lines
labeled on it), 2 LB/AMP plates (1 black line labeled on it), 1 LB/araC (2 black lines labeled on
it) and 1 LB/AMP/araC (1 red line labeled on it). On the bottom, label each of these plates with
your group’s name or number, the type of plate that it is, your lab TA’s name, and whether or not
the pGLO plasmid was exposed to the E. coli sample. After the ten minutes is up, remove the
foam tray and place it into a water bath, set at 42 degrees celsius, for 50 seconds. After the 50
seconds is up, immediately transfer the foam tray directly back into the ice bucket. Immediate
transfer allows for optimum transformation results to occur. Let the tubes sit in the ice bucket for
another two minutes. Remove the foam tray with the tubes from the ice bucket for the last time
and place the tray on the laboratory table. Open one of the tubes, grab a new, sterile pipette, extract 250 uL of LB nutrient broth and put it into the tube. Make sure to mix the LB nutrient
well with the tube’s solution and then close the lid. Using another new, sterile pipette, repeat this
step again for the other tube. After both tubes have received the LB nutrient, let the tubes sit at
room temperature for 20 minutes. Tap each of the closed tubes to mix the solution. Use a new,
sterile pipette and extract exactly 100 uL of solution from one of the tubes and put it onto the
corresponding petri dish. Repeat this step for both of the tubes, using a total of two new, sterile
pipettes (one for each). For each of the plates, use a new, sterile loop and spread the suspensions
evenly around the surface. Be sure to not press the loop too deep into the jar—just lightly brush
across the surface with the loop. Stack the plates in order, -pGLO/LB being on the bottom and
+pGLO/LB+AMP+ara being on the top, and tape them together along the sides of the plates.
Label the bottom of the stack with the appropriate group name/number and class period. Place
the stack upside down inside of the incubator. Check back for results after one day. The
independent variable in this experiment was the presence or the lack of the plasmid (pGLO). The
dependent variable in this experiment was the bacteria growth and the fluorescence of the
bacteria. The controls of the experiment were the sterile pipette, sterile loop, the ice bucket and
the water bath. The second row of petri dishes is a group that was successful in getting the GFP gene to be
expressed. The third row is the row of results for this experiment.
Results:
E. Coli Growth and Fluorescence
-pGLO
LB -pGLO
LB + AMP +pGLO
LB + AMP +pGLO
LB + ara (1) (2) (3) (4) +pGLO
LB + AMP
+ara
(5) Growth
Present
(+ if present
and - if not
present) + + - + - Fluorescent
Under UV
Light
(Actual) No Glow No Glow No Glow No Glow No Glow Fluorescent
Under UV
Light
(Prediction) No Glow No Glow Glow Glow Glow Discussion:
For the Bacterial Transformation experiment, the hypothesis was neither supported or rejected.
Although colonies were formed, the samples did not glow and the GFP gene was not expressed
in any of the colonies. Those colonies expressing the bla protein promoted growth, while those
repressing the bla protein died in the presence of ampicillin, and this held true for the
experiment. The expression of the bla protein, along with the presence of the ampicillin, only
partially supports the hypothesis. The samples that had the plasmid inserted into it did not
support the hypothesis since none of the cultures expressed the glowing trait. Possible causes for
the GFP gene being repressed might be because of a lack of plasma being inserted into the
sample (not enough) or from the timing being off enough to the point where the plasma was not
weak enough to get into the E. coli. The transfer from the water bath to the ice could have been
too slow to properly allow transformation. In any experiment, with human regulation, there is
always room for human error and that is what I believe caused the flaws in this experiment—I do
not believe it was a flaw within the experimental design itself. A future implication of this
experiment could eventually lead to the improvement of antibiotics, how to make disease/illness
glow within living organisms so that they can be targeted and cured in an appropriate manner,
and also to help scientists further understand the manifestation process of E. coli bacteria and
how to contain it. ...
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