The Use of Restriction Digest & Gel Electrophoresis to Identify a Plasmid Conferring Ampicillin Resistance to Bacteria

By:

Sonia Rahangdale

Team Uncle Jesse

LBS 145

Section M2

Dr. Luckie

4/29/05

Abstract

            This experiment involved working with antibiotic resistant bacteria. Antibiotic resistant bacteria are a developing problem in the world today. From excessive use and increasing dependency antibiotics are found everywhere and bacteria are developing resistance. Most bacteria acquire resistance through plasmids, which impart new functions to the bacteria they occupy and circulate between cells. Resistance commonly develops in areas where bacteria are continuously exposed to antibiotics. The areas where we swabbed for different bacteria include bathroom sinks, drains, and showers, where antibacterial cleaning products and hand soaps are regularly used.

In our experiment our goal was to determine what plasmid coded the bacteria we obtained for its resistance. We obtained bacteria and then placed them onto separate LB agar plates containing antibiotics. After three days, antibiotic resistant bacteria grew. A specific colony was removed and harvested in order to obtain more plasmid DNA which was then isolated through lysis. A restriction enzyme digest was performed, to fragment the DNA. The fragments then underwent gel electrophoresis and they separated on the agarose gel. By knowing the characteristics of the plasmid, such as its ability to confer resistance to ampicillin and determining the distances the fragments traveled and correlating them to the number of base pairs in the plasmid, the plasmid could be identified.

We found that our bacterial colony was resistant to ampicillin. Running a gel electrophoresis confirmed the presence of DNA in our plasmids and the restriction digest fragmented the plasmid DNA. Due to time constraints the second gel electrophoresis was not ran so the plasmid was not identified.

Figure 1a. These five plates were the control plates (no antibiotics in them) there was a large amount of bacterial growth which was expected due to the fact that the bacteria could grow without needing to be antibacterial resistant. The bacteria were collected from the Q-tip, the toilet, the shower drain, the compactor drain and the sink drain.

Figure 1b. These five plates contained ampicillin. All the plates produced growth, showing that the bacteria were resistant to ampicillin.

Figure 1c. The bacteria were exposed to tetracycline in these five plates. There was no growth in any, indicating that the bacteria were not resistant to this antibiotic.

Figure 1d. The bacteria from the five areas we swabbed are on plates with kanamyacin. There was growth on all of the plates indicating resistance.

Discussion

Hypothesis & Predictions

In an attempt to answer the question of whether or not there are strains of antibacterial resistant bacteria around Holmes Hall and if so what plasmid coded them for their resistance, an in depth investigation was ran. Before running any tests I hypothesized that there were indeed bacteria that was resistant to the effects of antibiotics and that a plasmid most likely coded it for its resistance. Also, it was thought that the bacteria would be resistant to ampicillin, kanamyacin, or tetracycline since these are commonly used antibiotics. This was hypothesized due to the fact that bacteria everywhere are gaining resistance and this is an increasing problem facing the medical world (Anonymous, 2005). Plasmid circulation is a common method of gaining resistance since plasmids can bestow new properties upon the cell which it occupies (Lewis, 2002). It was predicted that the specific plasmid that conferred resistance to the bacteria would be able to be identified through the extensive tests in the procedure.

Swabbing for Bacteria

After conducting this experiment it is clear that there are in fact antibiotic resistant bacteria in Holmes Hall. Bacteria are more likely to obtain resistance to antibiotics in a constrained environment where they are exposed to antibiotics on a regular basis (Tenebaum, 2005). Areas were swabbed where antibiotics are regularly used. The 5 areas of choice were: bathroom sink handle, bathroom sink drain, compactor room drain, shower drain and toilet seat. The swabs were placed in test tubes containing LB broth and after incubating them overnight there was no obvious bacterial growth. This could be because the areas were not properly swabbed and run over the surface enough, the swab was ran over the area once, possibly not picking up enough bacteria. Also, when swabbing the area. None of the swabs produced adequate growth, which indicates improper swabbing and not enough bacteria obtained initially before amplification. The second round of swabbing was over the same areas with one exception; rather than the bathroom sink handle, the swab itself was tested for bacteria as a control, each site was swabbed three times. This time around, all swabs produced growth and of the total 15 swabs the 5 with the most growth were chosen to test for resistance. Of the 20 plates the plate with the antibiotic ampicillin that was swabbed with bacteria obtained from the shower drain had the most growth. This is likely because showers are exposed to more antibiotics than any other site swabbed. From cleaning the shower itself and using antibacterial soaps on the body the shower drain is continuously exposed to antibiotics. All other areas exposed to ampicillin grew bacteria as well, the shower drain simply produced the greatest number of individual colonies, therefore it was chosen to continue experiments with. On plates treated with kanamyacin, the bacteria obtained from the toilet had an excessive amount of growth. This indicates that the antibacterial that the toilet was cleaned regularly with by Holmes Hall maintenance contained more kanamyacin than any other antibiotic. The same theory can be used to explain the growth on the shower drain plate treated with ampicillin; perhaps the shower is cleaned with antibiotics containing more ampicillin so resistance has developed.

Bacterial Growth

All other plates contained various amounts of growth from all areas swabbed with one exception; all plates treated with tetracycline had no growth whatsoever. This indicates that tetracycline is rarely used in common cleaning agents and therefore resistance has not developed against this specific antibiotic. The samples that were exposed to no bacteria at all had large amounts of growth due to the fact that all bacteria was able to grow, whether it had been coded to resistance or not.

Lysis & Gel Electrophoresis

The next steps were to take the colonies from the shower drain plate treated with ampicillin and harvest them to obtain more bacteria to work with and then lyse the bacteria to obtain plasmid DNA. Lysis removes the plasma membrane as well as double stranded DNA, leaving only plasmid DNA (Khra et al., 2005).

A baby gel was then ran to confirm the presence of DNA. The gel electrophoresis was ran on an agarose gel and then placed under UV light. This confirmed the presence of plasmid DNA as there were orange streaks running across the length of the gel where the plasmid DNA mixture was first loaded into wells. The presence of DNA was expected due to the fact that the bacteria were antibiotic resistant and the most common way a bacterium obtains resistance is through plasmid circulation (Lewis, 2002).

The gel electrophoresis does not confirm that the bacteria obtained resistance through the plasmids. Mutations can occur in chromosomal DNA which change the genetic material thereby making the bacteria resistant (Bren, 2002).

Restriction Digest & Gel Electrophoresis

In order to confirm that it was, in fact, a plasmid that coded the bacteria for its resistance a restriction digest was ran. The restriction enzymes BamH I and Hind III were used. These were used because they are known to cut plasmids that confer resistance to ampicillin (Khra et al., 2005). In order to identify the plasmid a gel electrophoresis would have to be run on the plasmid DNA that had undergone a restriction digest. Then the fragments of the plasmid would separate across the gel. By measuring the distances the fragments traveled and correlating them with the number of base pairs in a plasmid it is possible to identify the plasmid using plasmid maps. By adding all the lengths of the base pairs up a total number of base pairs for the entire plasmid could be found. Due to time constraints and having to repeat parts of the experiment such as swabbing, however, only the restriction digest was performed but the second gel electrophoresis was not.

Sources of Error

Errors throughout this experiment include the initial ineffective swabbing which did not produce enough bacterial growth and wasted time. During lysis when the supernatant had to be removed, leaving a bacterial pellet, errors could have been made. Human error in attempting to remove all the supernatant and none of the bacterial pellet could lack in precision. Also, contamination is a possible source of error. Bacteria are everywhere and leaving the lids of plates open for even a short amount of time could lead to contamination. Restrction enzymes, if not stored properly could work inefficiently or not at all at digesting the plasmid DNA. Also, our restriction digest was ran for too long of a time period, this most likely resulted in the restriction enzymes cutting the plasmids properly along specific base pairs; yet after this occurred the enzymes could begin to cutting in random places because the digest was ran for too long. Running the digest too short will result in the plasmid DNA not being properly fragmented; yet running it too long will result in meaningless fragments. Improper digest would lead to an unidentifiable plasmid.

Although a specific plasmid coding the bacteria for its resistance was not found in this experiment, the presence of antibiotic resistance bacteria around Holmes Hall was confirmed. This supports the initial hypothesis and confirms the prediction that antibiotic bacteria were in fact common enough to be found in and around Holmes Hall. This ever growing problem needs to be addressed and more care needs to be taken in using antibiotics. If changes are not made antibiotics, which were once hailed as a wonder drug that saved lives could become as useful as a placebo (Bren, 2002).