ATTEMPTED IDENTIFICATION OF AN ANTIBIOTIC RESISTANT BACTERIAL PLASMID USING GEL ELECTROPHORESIS

Written by: Rebecca Hurst

4/25/05

Abstract

The main focus of this experiment was to identify an antibiotic resistant bacterial plasmid. This was done to help increase the number of known antibiotic resistant plasmids. Bacteria were grown by swabbing off of Holmes Hall bathroom exiting doorknobs, soap dispensers, and hot water faucets in both men’s and women’s restrooms. Four replications of each area were done. The bacteria were grown in LB broth. Antibiotics were then added to each of the tubes. The antibiotics used were ampicillin, kanamycin, and tetracycline. One replication was used as a control, while one antibiotic was added to each of the remaining three tubes. Streaking was done to transfer the bacteria to antibiotic agar plates. Antibiotic resistant colonies were grown by incubating the plates for 24 hours. One colony was then divided and transferred into four separate tubes: two tubes containing the same antibiotic, and one each of the remaining two. This was done to replicate resistance. One colony was transferred to have a better chance of having one unknown instead of several. Alkaline lysis was used to extract out the plasmid DNA. A gel of the purified DNA was run to guarantee the presence of plasmid DNA (Krha et al., 2005). Harvesting and purification procedures were done several times to no avail. Had DNA been present, restriction digest would have been used to cut the DNA at specific sites. Gel electrophoresis would have been used to compare the unknown plasmid to the lambda ladder and then a known plasmid of similar properties.

(a) Control plates from both girls’ and boys’ restrooms

(b) All plates

Figure 2: It can be seen that while the boys’ restroom contained more bacteria, the girls’ restroom grew more antibiotic resistant bacteria. (a) Control plates showing more growth from boys’ restroom. (b) All plates shown; plates with pink dots in center are the only plates with significant growth other than the control plates. Both of these plates are from the girls’ restroom.

Discussion

For this experiment, we swabbed three different areas of the boys and girls bathrooms in East Holmes Hall. The places swabbed were the door handle leading out of the restroom, the button on the soap dispenser, and the hot water handle on the sink. Four replications were performed on each surface, in each restroom, leading to a total of 24 swabs. After several attempts at harvesting and plasmid DNA extraction, no valid plasmid DNA was found. Had DNA been found, restriction digests and gel electrophoresis would have been used to produce a plasmid map for the unknown plasmid.

It was predicted that the bacteria grown on control plates would have lawn growth. This is because there are no antibiotics present in theses plates to kill off any bacteria. This prediction was supported (Figure 2a). We also predicted that the boys’ restroom would show more bacterial growth than the girls’ restroom. This prediction was also supported (Figure 2a). It was predicted that the girls’ restroom would contain more antibiotic resistant bacteria. The plates containing antibiotics and bacteria from the girls’ restrooms were the only non-control plates that show noticeable bacterial growth (Figure 2b).

We expected to find colonies on the plates containing antibiotics that the bacteria were resistant to. We isolated this colony and allowed it to replicate over night by vigorous shaking and incubation. After the antibiotic resistant bacterium was isolated, DNA extraction by alkaline lysis was performed (Krha, et al., 2005). This was done to extract and purify the plasmid DNA. After this step, a gel was run and observed under UV light to ensure the presence of plasmid DNA. The loading dye should have bound to the DNA producing a glowing band. However, after several failed attempts at the extraction and confirmation of the presence of plasmid DNA, the possibility of creating a unique plasmid map became highly unlikely.

Had plasmid DNA been extracted, more steps would have been taken in order to produce the unique plasmid map that we originally hoped to create. Restriction digest would have been performed on the unknown plasmid, cleaving it at certain sequences, in order to run the plasmid DNA on a gel. Approximately 2 restriction enzymes would have been used so that the pieces would have been small enough (no less that 500 base pairs) to move through the gel. A lambda ladder would then be run alongside the unknown plasmid DNA in gel electrophoresis in order to estimate the lengths of the sequences in the unknown plasmid DNA. After, another gel would have been run, with the lambda ladder, the unknown plasmid DNA, and a known plasmid cut with the same enzymes used in RE digest. After performing all of these steps, a plasmid map would have been produced.

After extensive research, it was concluded that there are several explanations for why no plasmid DNA was found. The most obvious reason does not need to be explained by research, however. This reason is simple human or mechanical error. Although extreme precaution was used when performing the lysing procedure, there could have been errors made. Preciseness may have not been achieved considering the pipette malfunctions that could have occurred. Although the failure of lysing was most likely the reason for no DNA being found, other research was still done.

Mutations in a ribosome or protein that can change the site where an antibiotic binds can also cause antibiotic resistance in bacteria. Both tetracycline and kanamycin bind to a specific region of a ribosome in order to interfere with protein synthesis (Criswell, 2004). Other ways that bacteria resist the effects of antibiotics are as follows: alteration of the outer membrane structure preventing the antibiotic from entering, modification of binding enzymes, synthesis of beta-lactamases that cleave the functional lactam ring of the drug, alteration or degradation of the drug once it enters by synthesis of special enzymes, and finally the simple natural resistance of some bacteria to certain antibiotics (Bauman, 2003).

While performing all tests, it was extremely important to be precise and know what the methods are ahead of time. For instance, during the extraction of plasmid DNA, it was necessary to add exact amounts of the solutions to ensure proper DNA retrieval. Extreme precaution was used when handling the ethidium bromide (a mutagen) during the gel preparations, and while performing the actual gels (using the correct voltage, etc.). As in any lab, there can also be inconsistencies from day to day with the machinery. Extreme precaution and good documentation was used to ensure accurate results.