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.
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.
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.
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
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.
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Purves, W.K., G. Orians, C. Heller, and D. Sadava. 1998. Life The Science of Biology, 5th ed. Sinauer Associates, Inc., Sunderland, MA.