Kanamycin Resistant Bacteria in Red
Cedar Water; Gel Electrophoresis and Restriction Digest Lead to Unknown Plasmid
Jessica Grover
LBS 145: Cell and Molecular Biology
Section M1 Lab
Rebecca DeGraaf and James Hardie
Abstract
ÊÊÊÊÊÊÊÊÊÊÊ Plasmids have genes that code for the production of a protein which expresses antibiotic resistance.Ê Although antibiotics are helpful in destroying bacteria, their overuse and their capability to quickly evolve mechanisms for antibiotic resistance makes them damaging to the environment, especially water systems.Ê A question was asked as to what type of plasmids would be in bacteria from the Red Cedar River and if the bacteria were resistant to tetracycline, ampicillin, or kanamycin.Ê
ÊÊÊÊÊÊÊÊÊÊÊ Samples of water and LB broth were grown to detect the presence of bacteria, and then bacterial plates were grown with nutrient agar and antibiotics with a sample of water from each of the four sites.Ê The tetracycline plates and the sample from underneath the bridge exposed to kanamycin produced no bacterial growth.
ÊÊÊÊÊÊÊÊÊÊÊ When
a miniprep gel of seven lanes was constructed (
ÊÊÊÊÊÊÊÊÊÊÊ HindIII and AvaII were the restriction enzymes used.Ê When compared to the KB- Ladder in the first gel, the DNA provided bands (23,000, 5,000, 200, 90, 70 bp) to determine a plasmid to run in the second gel, which was inconclusive.Ê These procedures were expected to provide evidence to identify a plasmid from an area of water located on the Red Cedar River, however, the results were inconclusive.
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Figure 6: The above figure shows Gel I, which was a gel consisting of a KB- Ladder (A) with 3 restriction enzyme solutions: calm water resistant to ampicillin (B), calm water resistant to kanamycin (C), and before the rapids resistant to kanamycin (D) were all cut with AvaII and HindIII restriction enzymes.Ê There was DNA present in well B, however, the restriction enzymes did not cut well, which resulted in the large, faint pieces of DNA not being able to move down the gel.Ê The calm water resistant to kanamycin had bands of DNA that moved down the gel.Ê The sizes of these bands were 23,000 bp, 5,000 bp, 200 bp, 90 bp, and 70 bp.Ê The last solution with restriction enzymes and the sample of water taken before the rapids and resistant to kanamycin did not have distinctive bands of DNA and, instead, was viewed as a blurry clump of RNA.
Discussion
The purpose of
this experiment was to identify a plasmid of antibiotic resistant
bacteria.Ê The areas sampled for bacteria
on the Red Cedar River were the area of water off
Where the Water was Collected and Why
The samples of water were taken from the Red Cedar River because bodies of water are known as high traffics areas for bacterial growth (Reinthaler et al., 2003).Ê Since the Red Cedar is close to a water treatment plant, it was thought that bacteria would be found in the river due to contamination of groundwater which results from the circulation of treated water with remaining amounts of bacteria still present throughout the environment (Reinthaler et al., 2003).
Antibiotics and Resistance
All three
antibiotics, tetracycline, ampicillin, and kanamycin, were used for the
experiment to serve as replications for bacterial growth against more than one
type of antibiotic.Ê Ampicillin was of
particular interest because high resistance rates exist for ampicillin and
antibiotics within the penicillin group (Reinthaler et al., 2003).Ê In fact, the solutions exposed to ampicillin
had the most amount of lawn growth.Ê
Rather, they had bacterial lawn growth spread about the plates, which
presented an idea that the bacteria were resistant to ampicillin.Ê The kanamycin plates mostly had colony growth
and appeared to be unorganized.Ê It also
showed that the bacteria were resistant to the kanamycin.ÊÊ
Gels and Their Results
The goal of the experiment was to identify the name of the plasmid from bacteria that was resistant to an antibiotic.Ê The picture of the miniprep gel with seven lanes was very faint and does not clearly show the presence of DNA in the three lanes it was found to be present.Ê However, there was DNA present in the three lanes.Ê To prevent the bands from being faint in the actual gels with restriction enzymes, the concentration of DNA was raised to produce a brighter band.
The plates did not show growth in the tetracycline, and ampicillin resistant solutions showed growth but did not appear on the first gel.Ê Therefore, the unknown plasmid was predicted to be from bacteria resistant to kanamycin because it grew on the plates and appeared in the gel.Ê Since the DNA found in the final gel was still visible in the well and did not travel down the gel, it was concluded that the DNA was genomic, and it was just too big to move down the gel to produce bands (Werner, 2005).Ê The DNA was not plasmid DNA because plasmid DNA is smaller and would have been cut by restriction enzymes and yielded bands of DNA.Ê Also, only 10 plasmids were shown in the course pack, and there are many other ones that exist, so it was very rare that a plasmid would have been identified.
It is possible that the gel was not run long enough or the voltage was set too high at the beginning.Ê The voltage for the final gel was set at 96 volts instead of gradually increasing the voltage from 84 to 96 volts for the last half hour.Ê The voltage, the agarose concentration, or the strength of the ions determines the way the DNA fragments move down the gel (Martin, 1996).Ê If the DNA was not genomic, large plasmids would have been present, therefore, a lower concentration of the agarose gel would have been necessary for the large fragments to be digested and allowed to move farther down the gel.Ê The concentration for the gel was 50 ml of buffer and 0.4 grams of powdered agarose, but since the DNA did not move down the gel, the concentration should have been reduced to allow the fragments to travel.Ê Supercoils, double stranded, circular DNA molecules, may be another explanation for why faint bands appeared on the first gel and have a base pair size of 2000 to 50000 bp (Martin, 1996).Ê Supercoiled particles can be affected by the amount of ethidium bromide in the solution as well (Martin, 1996).ÊÊÊÊÊ
Errors
ÊÊÊ It is possible that some types of error may have occurred while carrying out the experiment.Ê Cross- contamination may have happened due to the fact that other solutions, such as Solutions I, II, and III used in isolation, were used by the entire class.Ê Perhaps the water samples were not kept at a consistent temperature while being transported from location to location, or the test tubes were contaminated.Ê Considering the majority of the growth on the antibiotic plates was lawn growth, a highly unusual event, the results may have been affected.Ê The random, individual colonies swabbed with an inoculating loop may have had more than one type of colony on it.Ê If this occurred, then more than one plasmid would have been amplified resulting in a solution of different plasmids and no way of identifying a single one.Ê It is possible that while streaking the plates with the LB broth and bacteria, the inoculating tube may have contracted other bacteria from the surrounding air and produced bacterial growth not from the Red Cedar, which may have affected the results of the experiment.Ê
Another source of error may have occurred in the purification process.Ê The bacterial pellet may not have been dried long enough, the supernatant may not have been separated from the pellet, or some of the pellet may have been pipetted into the supernatant accidentally.Ê It is likely that perhaps the TE buffer did not contain enough RNase, which may have been the cause of the faint DNA bands on the miniprep gel, otherwise the bands would have been brighter.Ê Maybe the gel needed to be run for a longer time in order for the fragments to be efficiently distributed along the gel and present more defined and bright bands.Ê Although DNA Strider was used to determine the most accurate combination of restriction enzymes to use on the DNA sequences to cut the strand of DNA, HindIII and AvaII may not have been the most efficient pair.Ê This is due to the fact that there were only 7 enzymes provided, and out of those seven, the two that were chosen may have worked even better with a different enzyme.Ê There are many other enzymes that could have been used, however, the supply was limited to seven.Ê
Another source of error that may have been a factor in the results was pipetting the DNA solutions with loading dye into the wells of the gels.Ê Maybe some DNA leaked out or a hole was punched through the bottom of the well and wasnât seen.Ê Also, the pipettes are not calibrated to pipette one microliter of solution.Ê So the final concentrations of solutions with DNA and enzymes may have been altered.Ê When replicating the bacteria from the plates in LB broth, catenanes, interlocked rings of plasmids, may have been created (Hardy, 1986).Ê This would have prevented their movement down the miniprep gel if they werenât purified before gel electrophoresis.Ê This can be seen in the miniprep gel in the other 4 lanes that did not reveal the presence of DNA.
During isolation and purification of the DNA, some chromosomal DNA may have contaminated the plasmid DNA, or the plasmids may have broken.Ê It is possible for chromosomal DNA to contaminate the plasmid DNA during isolation and purification because the pellet may have been disrupted by the pipette and resulted in a little mixing in with the supernatant.Ê As explained by Hardy, when cells were broken open to remove the debris and unnecessary contents, some plasmids may have been broken while being centrifuged, which could have resulted in faint or unseen DNA bands on the gels (Hardy, 1986).