Genotoxicity: Coffee
Abstract: With this experiment, our primary goal was to determine whether the mutagen we used had sufficient changes in our Salmonella Typhimurium's DNA to consider it a mutagen. The results our specific group ended with were unusable and thus we obtained data from a separate group’s incubation dishes. The colonies the coffee stained Petri dish created were around 45 and we ended with a reversion frequency of 2.25×10^-7.
Introduction: The chemical we chose to test for our experiment was coffee. Often, coffee is one of the top listed amongst everyday products that can be viewed as controversial for their possibility of being carcinogenic. According to the IARC in 2016, there continued to be insufficient evidence that would show whether or not drinking coffee was a factor in inducing cancer, however, this uncertainty continues to flood the questions and research platforms on what effect coffee actually has on human health. Coffee tends to include the chemical Acrylamide, which is often used in industrial processes. This is the main concerning factor in the carcinogenic doubtfulness of this popular drink. In fact, in California, this chemical is one of 65 that a recently established law requires to carry a warning alongside for its possible association with cancer. According to the American Cancer Society, Acrylamide is formed as a byproduct of amino acids and sugars in starchy foods being raised to high temperatures and although is not 100% correlated to being a human carcinogenic, they have seen such effects in animal testings. Studies continue to show inconsistent results and there is varying data between effects on types of cancer as well as sex and other varying factors. The goal of our research and our experiment was to determine whether or not we have a carcinogenic mutagen in coffee. As a group, we hypothesized that coffee would affect mutations amongst our Salmonella DNA dish and we would expect to see more colonies in the experimental Petri dish than in our control dish. The Ames test allows us to observe this phenomenon in a faster than usual time frame by allowing us an environment in which the metabolic needs of bacteria help emphasize whether mutations have occurred or not.
Methods: In our experiment, we first started by completely cleaning our work area off and putting on goggles and gloves since we were dealing with biohazards. In the first exercise we conducted, our goal was to isolate genomic DNA. We started this process by pipetting 1 mL of water to a microcentrifuge. We picked one colony off of the plate that contained the experimental mutagen using a wooden swab and completely suspended the colony into this sterile water. Next, we made sure our centrifuge was balanced and added our tube for 2 minutes at 6,000rpm. We then removed the supernatant, which is defined by Oxford’s Dictionary as, “the liquid lying above a solid residue after crystallization, precipitation, centrifugation, or other process”, using a p1000 and discarded it in a waste beaker. Then, we added 200 microliters of InstaGene matrix to the pellet and incubated it for 20 minutes at 56 degrees Celsius. We vortexed the tube every 5 minutes to keep it in suspension. The InstaGene matrix is a commercially available kit to isolate genomic DNA. It is also made with 6% w/v Chelex resin that makes DNA sample preparation easy and cost-effective so that PCR-quality template DNA can be provided in less than an hour. After incubation, we vortexed our tube at a high speed for 10 seconds then placed it into a boiling hot water bath for approximately 8 minutes. Once the 8 minutes was up, take the tube out and vortex it again for 10 seconds at a high speed then spin at 6,000rpm at 6 minutes. After this, we pipetted the supernatant with a p1000 and placed it in a fresh micro-centrifuge tube. In experiment 2, we brought our tube with the supernatant, which contains the genomic DNA, that we collected from the first experiment and transferred 20 microliters of the supernatant into the correct well on the 96 well plate. We then added 20 microliters of PCR master mix to our well. The PCR master mix contains primers, buffer, dNTPs, Taq polymerase, and Magnesium ions. After, we gently used a pipette to mix our solution by moving the pipet up and down the solution. Finally, we gave our TA the samples so they could load our solution into the thermal cylinder and begin this reaction.
Results and Figures
Colony Growth in Positive Control Plate, Experimental Plate, and Negative Control Plate
Figure A Figure B Figure C
Positive Control Plate Experimental Plate Negative Control Plate
(Figure A, Figure B, Figure C: Jenny Johnson, 12:30-3:20 lab, Kana)
Results: Growth of colonies were present in the positive control plate (Figure A), indicating the mutagen coffee was present and reacted with S. typhimurium. The presence of colonies of bacteria in the culture indicated that the positive control was successful, expressing random mutations. Colony growth in the experimental plate (Figure B) indicates coffee is a possible mutagen. Growth of colonies was not present in the negative control plate (Figure C), indicating the negative control was successful, with no mutations present.
Qualitative Data of Coffee on Bacteria
The positive control plate (Figure A) presented a growth of five colonies. These colonies were small, but proved the test to be successful.
The experimental plate (Figure B) also presented a growth of five larger colonies.
The negative control plate (Figure C) presented no colony growth.
Quantitative Data: Reversion Frequencies
The potential mutagen, coffee, was calculated to have a reversion frequency of 2.25 x 10-7. The positive control had a reversion frequency of 2.18 x 1010. The negative control had a reversion frequency of 9.2 x 109.
Other Results?
Discussion: Our evidence that there was more colony growth on our positive control plate (figure A) than our negative control plate (figure C), supports that our positive control, 4NOP, caused a mutation in the TA 1538 strain to promote bacterial growth (Goodson-Gregg and De Stasio, 2009). On the negative control plate, sterile water was used as our mutagen, which supports evidence that there was the least amount of colony growth on this plate since water is not considered a mutagen and therefore was our negative control. Since the reversion frequency of our experimental plate was higher than the reversion frequency of our positive and negative controls, it supports that coffee is a significant mutagen to the TA 1538 strain of S. Typhimurium.
In the case of our experiment, the rate of the spontaneous mutation (coffee) was higher than that of our induced mutation (positive control). The spontaneous mutation rates of natural elements varies, however it has been found that the average rate is anywhere from 2-12*10^-6 mutations per gamete per gene. Also, it has been found that large genes mutate more frequently than smaller ones (McClean, 1999).
Furthermore, there is a difference in the rates and types of mutations based on different chemicals. For example, in this experiment alone there were two possible chemical strains to be used in the TA1535 and TA1538. The TA1535 strain contained a missense mutation that led to an amino acid substitution while the TA1538 strain was a frameshift mutation that led to two amino acid changes and a premature stop codon. Therefore, the chemicals used in these different strains resulted in different types of mutations and in this case, the rate of mutation is affected greater in the TA1538 strain due to the fact that it was altered more.
In conclusion, this experiment helped determine whether or not our chemical was mutagenic and therefore with further research can determine if our mutagen, coffee, is carcinogenic. Since the coffee plate had more colonies than our positive and negative plates, coffee is considered to be genotoxic. Also, our results can help us conclude that different mutagens such as coffee can be used to potentially change the phenotype of an organism just as it helped the his+ phenotype revert in S. typhimurium.