FAILED ATTEMPTS TO IDENTIFY A BACTERIAL ANTIBIOTIC RESISTANT PLASMID FROM BATHROOMS USING GEL ELECTROPHORESIS

By:Ê Lubna Ansari

Abstract

ÊÊÊÊÊÊÊÊÊÊÊ For this experiment, our end goal was to locate and identify an unknown bacterial antibiotic resistant plasmid.Ê We began by swabbing unknown bacteria off of the inner doorknob, soap dispensers, and the hot water sink handles of the East Holmes Hall boyâs and girlâs bathrooms.Ê There were four replications done on each area.Ê Then, we placed the bacteria in LB broth, were it reproduced overnight.Ê Bacteria grew in all the tubes.Ê The next day antibiotics were added; this was done to establish resistance.Ê The antibiotics used were kanamycin, tetracycline, and ampicillin.Ê The bacteria were moved to antibiotic agar plates.Ê The plates were incubated for 24 hours, which allowed for the bacterial colonies to grow.Ê All the control plates had plenty of bacterial growth.Ê The antibiotic plates with a considerable amount of growth were the girlâs doorknob with kanamycin, the girlâs doorknob with ampicillin, the girlâs soap dispenser with tetracycline, the girlâs hot water sink handle with kanamycin, the boyâs soap dispenser with ampicillin, the boyâs hot water sink handle with ampicillin, and the boyâs hot water sink handle with kanamycin.Ê We took off one colony from each of the plates because that gave us a better chance of acquiring only one plasmid.Ê We used the alkaline lysis method to extract out our plasmid DNA.Ê Then, we ran a gel on our DNA after purification to guarantee that DNA was present.Ê However, after performing the above steps three times using different bacterial colonies, we found no DNA.Ê Therefore, our end result was inconclusive.ÊÊ

Figure 1.Ê Visual of Gel Showing RNA.Ê The above picture shows that DNA extraction and purification did not work; only RNA was found, no DNA.

Discussion

ÊÊÊÊÊÊÊÊÊÊÊ In this experiment, we collected bacteria from bathrooms in order to eventually find and identify an antibiotic resistant plasmid.Ê We swabbed three different areas from both the boyâs and girlâs bathrooms in East Holmesâ Hall.Ê The three different areas were the exiting door handle, the ãpushä bar on the soap dispenser, and the hot water handle on the sink.Ê We did four replications of each, which totaled to 24 swabs.Ê We added three different antibiotics- one per each replication; the antibiotics used were tetracycline, amplicillin, and kanamycin.Ê No antibiotics were added to the 6 swabs, because they were used as controls.Ê We isolated the bacteria by allowing them to grow on agar plates.Ê Afterwards, we harvested the bacteria and performed lysis by alkali.Ê After performing DNA extraction and running the gel, we found no DNA.Ê Though our ultimate goal was to identify the plasmid and create a plasmid map, we also wanted to find out the types of antibiotic resistant bacteria we are exposed to every day- and where they are vastly located (i.e. the door handle, soap dispenser, and sink handle).Ê We were unable to do either.Ê However, we did find out which areas in the bathroom contained bacteria and which areas contained the most antibiotic resistant bacteria.

Similar experiments have been done- one such experiment was done by students from Miami University.Ê They wanted to compare the amount of bacteria between the men and women public bathrooms.Ê Their research allowed them to hypothesize that the womenâs bathroom would contain more bacteria due to the fact that women tend to use the public bathroom facilities more often then men (Davis, 2002).Ê Another experiment conducted by students from the same university was that they tested the amount of bacteria found on toilet seats vs. stall locks vs. sink handles vs. inner door handles.Ê They found that the inner door handles contained the most bacteria followed closely by the sink handles (Cisar, 2002).Ê Our predictions based off the previous experiments were that the inside door handle would contain the most bacteria, and then the hot water handle, and the soap dispenser would be a close third.Ê Our actual results showed us that bacteria grew in all three areas, but we couldnât differentiate where it grew the most.

It is known that many people donât practice appropriate hygiene, especially when it comes to hand washing.Ê The United States Centers for Disease Control and Prevention (CDC) estimated that one in three people do not wash their hands after using the bathroom.Ê But there are some people who do wash their hands using the antibacterial soap in our Holmesâ Hall bathrooms.Ê We predicted that the hot water handle and soap dispenser would contain more antibiotic resistant bacteria than the door handle.Ê This is because the soap contains antibacterial components that would make the existing bacteria resistant.Ê We, also, predicted that after adding the antibiotics to our bacteria none of the bacteria from the door handle will survive.Ê Therefore, the hot water handle and soap dispenser would be more resistant.Ê We found that the most antibiotic resistant bacteria were found on the exiting doorknob, then the soap dispenser, and lastly the hot water sink handle.Ê This could be because as people wash their hands and use the same hands to open and close the door, the antibiotic resistant bacteria attach to the door handle.Ê The more people touch it, the more the antibiotic resistance bacteria grow and thrive.

There are many reasons for why we were unable to acquire DNA.Ê Since this lab is all about precision, something could have been miscalculated or the DNA could have accidentally been thrown out.Ê Another experiment done with Bacteriophage Lambda replication discussed when lytic and lysinogen can switch of gene expression (Taylor, 1998).Ê In our case, the lysing process could have been too strong for our bacterial plasmids to survive.Ê There could have been problems with the DNA purification.Ê A article that discusses plasmid DNA- preparation stated that the problems could have been due to contamination by phenolic compounds and polysaccharides.Ê Also, it said that the best way to prepare a plasmid is to carry out the procedure without refrigeration- and throughout the experiment things were refrigerated daily (Betiku, et al., 2003).

To move this experiment further, one could keep trying till they get DNA and then move on to perform restriction enzyme digest and gel electrophoresis in order to attain a plasmid.Ê The restriction enzyme digest would help ID our plasmid because the enzymes cleave at certain sequences.Ê This would help is identifying the plasmid when comparing it to a known plasmid.Ê The gel electrophoresis would enforce the restriction digestâs results.Ê In the end, one would be able to identify the plasmid and create a plasmid map.ÊÊÊÊ