Abstract
Purpose of the experiment was to transform E. Coli into an antibiotic
resistant bacteria. A particular DNA sequence that encodes for resistance
to the antibiotic ampicillin was added to the bacteria E. Coli. Prior
to the experiment this resistant gene had been placed within a plasmid
which, for the purpose of this experiment, became the transporting factor.
By subjecting the mixture of the enhanced plasmids and the E. Coli to rapid
changes in temperature, while in the presence of CaCl2, it is thought that
the membrane permeability of the bacteria is increased; thus allowing the
plasmid to enter the cell more easily. Once inside the cell, mechanisms
of the cell transcribe the DNA from the plasmid, and, accordingly produce
subsequent offspring that are resistant to ampicillin. The first
part of this experiment involved the rendering of E. Coli resistant to
ampicillin. The second part of the experiment was placing unaltered
E. Coli and resistant E. Coli separately in either an environment that
lacked ampicillin or contained ampicillin. It was expected that cells
containing the gene would survive when in the presence of ampicillin.
This, though, was not the case.
Introduction
In this experiment a DNA fragment that encodes for the resistance to
the antibiotic ampicillin was transferred into E. Coli bacteria—creating
a transgenic organism. The host cell, in this case the E. Coli, transcribes
the new DNA and makes a protein that expresses this added feature.
The simplest type of vector, a plasmid, is the molecule that carries the
DNA sequences into the E. Coli. It is not understood exactly how
a plasmid enters a host cell, but in this experiment it was necessary that
Calcium Chloride (CaCl2) be present and that the cells undergo a shock
treatment—a rapid change in temperature from clod to hot and back to cold
again. The placing of non-resistant bacteria in an ampicillin rich
environment will cause the bacteria to be killed. But if bacteria
containing the ampicillin resistant gene is placed within an ampicillin
rich environment the expected results are that the bacteria would survive
and indeed grow. The antibiotic ampicillin is designed to destroy
E. Coli whose DNA sequence has not been altered by the addition of a gene
that is resistant to ampicillin. It is expected that cells containing
the gene that codes for resistance ampicillin will survive and grow in
an environment where ampicillin is present.
Materials and Methods
The wearing of gloves and eye-goggles was essential for this experiment
because the possible harmful effects of the bacteria E. Coli. Two
test tubes were marked; one labeled -AMP, and the other labeled +AMP.
Added to the two test tubes was 250ul of CaCl2—then both were placed on
ice. A loop full of the bacteria E. Coli was placed into each test
tube, and after the contents were mixed, by flicking the tubes, the tubes
were returned to the ice for 5 minutes. The gene for resistance to
ampicillin was added to the test tube labeled +AMP by using a plastic loop
full of the substance. The test tubes were then left on ice for 15
minutes.
Four agar plates were labeled as follows: 1) Negative AMP, Positive
Gene, 2) Negative AMP, Negative Gene, 3) Positive AMP, Negative Gene, and
4) Positive AMP, Negative Gene. Next, the two tubes were submerged
into a hot bath with a temperature of 42 degrees Celsius for 90 seconds
after which point they were removed and replaced back into the ice for
1 minute. After a minute had passed 250ul of the LB broth was added
to each test tube, and the contents were mixed about. A 100ul drop
of the solution from the test tube labeled +AMP was placed on two of the
agar plates labeled +AMP, +Gene, and -AMP, +Gene. This step was repeated—a
100ul drop from the -AMP test tube was placed on the remaining two agar
plates labeled -AMP, -Gene, and +AMP, -Gene. Next a glass rod was
used to spread the bacteria in each of the agar plates. Between each
spreading the glass rod was dipped in ethanol and lit on fire—a few seconds
were allowed for the glass rod to cool after the fire went out.
Parafilm was placed around each agar plate and they were then left
undisturbed for 1 week. A week later the plates were checked and
the results recorded.
Results
Table 1 shows the results gained from this experiment. The agar
plate (plate number 1 in Table 1) that contained the gene resistant to
ampicillin but lacked the antibiotic ampicillin tested positive for bacteria—therefore
bacterial growth was observed. Plate number 2—which contained neither
ampicillin nor the ampicillin resistant gene—tested negative. Plate
number 3, which harbored both ampicillin and the ampicillin resistant gene,
tested negative. No bacterial growth was observed in plate number
3. Lacking the ampicillin resistant gene, but not the ampicillin,
plate number 4 tested negative as well. Thus bacterial growth on
this plate was not observed.
Table 1
Observed
Plate # Gene and Ampicillin Status
Results
1 Negative AMP (-amp), Positive Gene (+gene) Tested positive
2 Negative AMP (-amp), Negative Gene (-gene) Tested positive
3 Positive AMP (+amp), Positive Gene (+gene) Tested negative
4 Positive AMP (+amp), Negative Gene (-gene) Tested negative
Discussion
Plate 2, as seen in Table 1 of the results, was the control of the
experiment because the bacteria was not altered from its original state—no
ampicillin resistant gene was added to the bacteria—and the bacteria was
allowed to grow in an environment to which it was suited. The absence
of the antibiotic ampicillin permitted its growth. If the antibiotic
was present, as is the case with plate number 4, then the expected results
would be no growth because the unaltered bacteria would be killed by the
antibiotic ampicillin.
The agar plate number 1, which had been altered by the addition of
an ampicillin resistant gene, tested positive simply because the antibiotic
ampicillin was not present. With the absence of the antibiotic the
bacteria was capable of growth. The fact that the bacteria contained
genetic information which made it resistant to ampicillin was insignificant.
The results for agar plate number 3 seem to have come out opposite
to what was expected. The addition of the ampicillin resistant gene
should have permitted some growth of the bacteria in the presence of the
ampicillin. The results in Table 1 show that no bacterial growth
occurred, thus an error must have taken place somewhere during the procedure.
It is likely that the error happened when using the glass rod to spread
the bacteria once it had been placed on the agar plate.
References
Vodopich, D.S. and R. Moore. 1996. Biology Laboratory Manual,
4th ed. Wm. C. Brown Publishers, Dubuque, IA.
Purves, W.K., G. Orians, C. Heller, and D. Sadava. 1998.
Life The Science of Biology, 5th ed. Sinauer Associates, Inc., Sunderland,
MA.