Using Kanamycin Resistance Bacteria to find the Sources of Contamination of Three Chicken Farms
Introduction:
Kanamycin is a common antibacterial that interferes with bacterial growth, by inhibiting protein synthesis, and causing the mistranslation of mRNA. Kanamycin is commonly used in chicken feed to keep harmful bacteria from getting into the eggs and producing healthier chickens. Recently reports of severe gastroenteritis have been linked to eating raw or undercooked eggs. This has led to the FDA to look for possible sources of contamination. Scientists have now isolated bacteria from batches of eggs known to cause the illness, and they found that the bacteria are resistant to kanamycin. The contaminated eggs were found to have
…show more content…
Each lab bench will make, and run one gel electrophoresis per table. Once the gel is ready to be loaded, load five microliters of PCR DNA ladder into the first well, as a standard. This should be found in a tube in and ice bucket. Next add two microliters of 6x loading dye into the six sample tubes. The dye should be mixed in thoroughly by gently pipetting up and down after adding the dye. Following that you should load fifteen microliters of each sample into the following six wells. Since lane one will have the DNA ladder lane two starts the samples using the orange tube, then the blue, yellow, red, green, and pink tubes go into lanes three, four, five, six, and seven respectively. Once all the samples are loaded turn on the electrophoresis machine, and wait until the bromophenol blue tracking dye has migrated at least half the length of the gel. Lastly using gloves carefully remove the gel and carry it to the UV light box to view, and photograph the gel (Hass C., Woodward D., and Ward A., 2010.).
Results:
The results of this experiment show that the farms do not share the same plasmid that carries the antibiotic resistance gene. Table one shows the individual group data for the concentration and frequency of the antibiotic resistant bacteria. Table two shows the overall frequency of antibiotic resistant bacteria for code A which was taken from Acme Farm, for the section.
As predicted the E. coli colony transformed with either the PUC18 or the lux plasmid developed an ampicillin resistance. Which made it easier for them to not only survive but also replicate in both the LB agar plates and the LB ampicillin rich agar plate. However the E. coli colony not treated with the plasmids could not survive and colonize in the LB ampicillin rich agar plates. The plate that had no ampicillin in its environment and no plasmid treated E. coli served as a positive control for this experiment because it demonstrated how the E. coli would colonize and grow in a normal setting. The cells in the positive control plate grew into lawn colonies because they were not placed into a selective environment or transformed, so they had no need to acquire ampicillin resistance. Two plates in the experiment contained E. coli cells that were transformed with either the PUC18 or the lux plasmid but were placed in an ampicillin free environment. These two colonies grew
I inoculated a T-Soy agar with bacteria number 118, for this I used a streak isolation method. Next, in order to distinguish between Gram positive and Gram negative I used a streak isolation technique on a CNA plate, then performed the same exact procedure on a MacConkey plate. The results from the CNA plate showed the Gram Positive bacteria was an Alpha hemolyzer. Next, I used a P Disc on a T-Soy agar inoculated with bacteria 118 and determined the Gram Positive bacteria was not sensitive to P Disc antibiotics. This revealed the Gram Positive bacteria to be Streptococcus Mitis. The results from the MacConkey plate proved the Gram Negative bacteria to be a lactose fermenter. With the Gram Negative bacteria I performed a lysine test with positive results. Next, I performed an ornithine test on the Gram Negative bacteria, with negative results, therefore I concluded the Gram Negative bacteria was Klebsiella pneumoniae.
This experiment was designed to test and observe the transformation efficacy of the pUC18 and lux plasmids in making E. coli resistant to ampicillin. Both plasmids code for ampicillin resistance, however, the lux plasmid codes for a bioluminescence gene that is expressed if properly introduced into the bacteria’s genome. The E. coli cultures were mixed with a calcium chloride solution and then heat shocked, allowing the plasmids to enter the bacteria and assimilate into the bacterial DNA. The plasmids and the bacteria were then mixed in different test tubes and then evenly spread onto petri dishes using a bacterial spreader, heating the spreader between each sample to make sure there is no cross contamination. Each of the dishes was labeled and then incubated for a period of 24 hours. The results were rather odd because every single one of the samples grew. Several errors could have occurred here, cross contamination or possibly an error in preparation as every single sample in the class grew, meaning all samples of the bacteria transformed and became ampicillin resistant.
We were using a restrictive enzyme to cut the DNA into smaller fragments. For the restriction digest we pipet 4 micrometers of enzyme mix into the bottom of each of our colored tubes making sure to use a new tip for each sample. Next, we capped the tubes and mixed the contents by flicking the tube a little bit with our fingers. After we mixed the contents, we tapped the tube to make sure all the liquid would go to the bottom of the tube. Then, we put the colored tubes in the heating blocks and we let them incubate for 35 minutes at 37 degrees Celsius.
The idea behind Gel Electrophoresis is that we inject a slab of gel with the DNA we found at the crime scene. We then inject the same gel, next to the crime scene DNA, with suspect 1’s DNA and suspect 2’s DNA. We then send an electric current through the gel and wait for the results. The smaller molecules in the DNA will travel farther than the bigger molecules because the bigger ones will have difficulty making its way through the microscopic beads in the gel. After the separate bands appear in the gel, we stain it with a special chemical called Ethidium Bromide to give it a color under the blue
The proteins are also added to a Laemmli sample buffer in order to give each protein a negative charge so it is able to get pulled through the polyacrylamide gel. The next step is to put the gel into the electrophoresis module and to run it. It is run until the proteins have almost reached the bottom of the gel. A blue tracking dye is added to the Laemmli sample buffer in order to track the distance in which the proteins travel through the gel. If it is run for too long, the proteins will run off the bottom of the gel and it will mess up your results. Once the protein reach the bottom of the gel, the gel is stained in order to be able to see the individual bands of the different proteins. When the gel is stained, the protein distances will be able to be measured and compared. For a detailed procedure, refer to the Comparative Proteomics Kit I: Protein Profiler Module Lab Manual.
We placed the gel into the running chamber, and then we completely covered the gel with TAE. 3 microliters of loading dye was added to each tube; this would help distinguish the enzyme from the gel. As before, we tapped the tube on the table to mix. Then we carefully added each of the four samples into their own wells. A total of 33 microliters of each sample was poured into each well. Afterwards, we attached the positive and negative electrodes to their corresponding terminals on the power supply and gel box. We turned on the power to around 80 volts and waited 45-60 minutes for the loading dye to move down the gel approximately 6-8 cm. Finally, we were able to visualize the DNA in the gel and write down the
Coli. The first standard E. Coli has no resistance plasmid while the second strain contains a resistance plasmid with genes protecting it from ampicillin. This standard E. Coli and pAMP (plasmid-Ampicillin) E. Coli were each streaked across plates containing the antibiotic and containing growth supportive Lurithea Broth. The purpose of this lab was to test their growth in each medium. Our hypothesis was that while the ampicillin resistant E. Coli would show growth in both LB and LB-AMP plate, the standard E. Coli would only grow in the LB plate for it contains no resistant plasmids against the
The transformation of E. coli using plasmid DNA was a success. The positive control plate had a near lawn of blue colonies growing on the plate. This indicated that the E. coli cells took up the plasmid and became ampicillin resistant. The blue colonies formed because the cells were able to produce β galactoisdase and in presence of X-gal the colonies turned blue. There were light blue colonies formed near the edge of the plate. This could due to the lower concentration of X-gal near the edge of the plate so those colonies were not really blue. In addition, there were too many colonies to count so we estimated the transformation efficiency of the positive control to be around 2000 units/μg. On the other hand, the negative is shown in figure
After analyzing the data recorded for both the agar plates containing ampicillin and those that did not, it can be concluded that the data provides enough evidence to reject the null hypothesis. There is enough evidence to support the alternative hypothesis stating that there is a correlation between plasmids coding for an antibiotic resistant gene and bacterial growth in ampicillin. When a bacterial solution containing either pUC18 or the lux plasmid is transformed in an agar plate containing ampicillin, only those cells which took in the plasmid are able to survive and replicate, forming individual colonies. Not all cells are transformed though, the chances of a successful transformation were extremely low. In order to see which cell transformed the cells were tagged according to their plasmid, in the plates containing pUC18 the only
After electrophoresis was finished, my boss removed the gel casting tray from the running chamber. Then, she carefully transferred the gel to a DNA staining tray. My lab assistants and I were going to stain it so it would be easier to see the
Let the agarose powder sit in the buffer for a few minutes. The beaker is covered with plastic wrap an placed in the microwave. Microwave the solution slowly with boiling it. As soon as the solution starts to boil, take it out and carefully mix it with the hot gloves on and continue to heat the solution until it is completely clear. Once the solution is cooled, add 3µL of ethidium bromide stock to the solution and mix it by swirling it. The gel is poured into the prepared mould that is taped on the ends and eliminate any bubbles. Place the comb on the negatively (-) charged side. After the gel solidifies, remove the comb and tape and place the gel into the chamber. Gently pour TAE buffer over the gel as the gel should be completely covered by 2-3 mm of buffer. If air bubbles form, gently displace them with a disposable micropipette tip. Droplets of prepared loading buffer are placed on a piece of parafilm paper. The ladder will consists of only the buffer because it will work as a measuring device to compare the DNA samples. 10 µL from each of the DNA samples will be mixed with the loading buffer using the micropipette tip. The positive control will be consist of 10 µL of water and the droplet of loading buffer. The samples including the ladder and positive control are added to a separate well in the gel. Once all of the samples are loaded into their own
Today, the use of antibiotics is very common. The gene that causes antibiotic resistance typically arises naturally or from a mutation. When bacteria are exposed to antibiotics, the bacteria with antibiotic resistance genes within the plasmid survive; this is called selective advantage. This process of the survival of bacteria that are resistant to antibiotics is a method of natural selection, causing more antibiotic resistant bacteria to be reproduced. They are rapidly reproduced by binary fission, conjugation, transformation,
The polymerase chain reaction or PCR for short can be used to create many copies of DNA. This allows the DNA to then be visualized using a dye like ethidium bromide after gel electrophoresis. The process has been refined over the years, however the basic steps are similar.
typhi. The transfer of ACCoT-resistance of S. typhi isolates has been reported earlier from different parts of India. It has been reported that about 120 kb plasmid encodes resistance to A, C, T, and streptomycin in S. typhi isolates from Kolkata (1989-1990). This result, the investigation of R-plasmid contained in strains of S. typhi. AGE (Agarose Gel Electrophoresis) analysis elaborated the presence of a single plasmid that is of approximately 55 kb among the isolates. Thus present findings suggest that MDR S. typhi may arise from sensitive isolates by acquisition of multidrug resistance plasmid from antibiotic-resistant enteric