Mutagenesis Poster
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printed by www.postersession.com The Mutagenic Potential of Caffeine on Saccharomyces cerevisiae Greggory A. Perry Johnson State college Department of Science Caffeine has long been studied as a potential mutagen, and proof has been provided to support this. The objective of this experiment was to determine whether or not the Topcare Stay Awake caffeine pill could have mutagenic properties when introduced into Saccharomyces cerevisiae replication cycle. Saccharomyces cerevisiae was exposed to 0.01%, 0.02%, 0.03%, and 0.04% concentrations of caffeine and allowed to incubate on YPD, -tryptophan, and – isoleucine agarose plated media to determine mutagenesis activity. Unfortunately, the results were inconclusive. This may have been from the possibility that the incorrect strain of yeast was shipped for the experiment. This hypothesis can be supported by examining the results of the controls. However, given research done on caffeine and its potential to be mutagenic, it would be worth investing in an experiment similar to this one. The tryptophan lacking media produced one colony. The isoleucine lacking media produced no colonies, and one of these plates was infected with mold, as can be seen in figure 2. The number of colonies on the YPD plates confirms that caffeine is not toxic and a good candidate for this experiment as seen on Figure 3 The positive and negative controls both had significant growth on YPD, while minimal growth occurred on tryptophan and isoleucine lacking media. (1) Pamela A. Marshall. Winter 2007. Using Saccharomyces cerevisiae to Test the Mutagenicity of Household Compounds: An Open Ended Hypothesis-Driven Teaching Lab. CBE-Life Sciences Education. Vol:6 pp. 307-315. (2) E. Carmen Candreva, Deborah J. Keszenman, Enrique Barrios, Ulises Gelόe, and Elia Nunes. 1993. Mutagenicity Induced by Hyperthermia, Hot Mate Infusion, and Hot Caffeine in Saccharomyces cerevisiae. CANCER RESEARCH. Vol:53 pp. 5750-5753. (3) Christopher P. Selby, Aziz Sancar. 1990 Molecular Mechanisms of DNA repair inhibition by caffeine. Proc. Acad. Sci. USA . Vol:87 pp. 3522-3525. The number of mutated colonies on all plates including the positive controls were insignificant, which leads me to believe that the strain of yeast that was used for the experiment was not correct. This could help to explain why I had no results with the mutagen I chose. I am still curious about my compound, but further experiments with different strains, and also different concentrations of caffeine are needed to say anything conclusive. The fact that there was growth on YPD plates confirms that the compound is non-toxic at this dosage, which provides a good starting point for future experiments. This is a good example of why controls are needed. Without the controls, it would appear that caffeine had little to no mutagenic properties. Several studies have been conducted on caffeine showing various results, the previously mentioned study conducted by Candreva et al. documented caffeine as a mutagen, but with some correlation to temperature. At 54˚C the rate of mutation was much higher than at 30˚C (2). This suggests high heat is breaking down elements of the compound allowing it to be accepted as a base. A study on E. coli showed that caffeine has the potential to inhibit the mechanisms of DNA repair. Selby and Sancar demonstrated that caffeine impairs DNA repair much in the same way as ethidium bromide; it inhibits nucleotide excision repair by promoting nonspecific binding of the damage recognition subunit, (3) otherwise it traps the subunit that repairs damage in regions of the DNA that do not need repair. Given the knowledge that has been obtained on caffeine already, I believe it is very important to continue the research on this potentially harmful compound that is consumed in mass quantities daily around the world. The D-7 strain of Saccharomyces cerevisiae is used in genetic experiments for their tendency to spontaneously mutate, and the fact that yeast are one of the simplest eukaryotic cells. This provides a good opportunity to study compounds because the yeast will tend to mutate if the compound is mutagenic. Candreva et al. have shown that caffeine at a high enough concentration (3g/l) and high enough temperature is responsible for a 5-fold increase in mutation within yeast. The study was based on a product regularly drank in Brazil, Known as Hot Mate Fusion. This common drink was found not to be as mutagenic at room temperature (2). The product I chose to test was the TopCare Stay Awake pill. This contains 200 mg of caffeine and small amounts of other compounds, mostly dye. The molecular structure of caffeine is that of a purine, (See Figure 1below.) which makes up half of each base pair in DNA and RNA. This gives caffeine an enormous potential to be mutagenic given the similarities. The concentration I chose to use is much lower than in Hot Mate Fusion, but the purpose of this experiment was to determine if the recommended dosage of caffeine was mutagenic. ABSTRACT INTRODUCTION AND PURPOSE METHODS RESULTS CONCLUSIONS BIBLIOGRAPHY Molecular formula of suspected mutagen (caffeine) in TopCare Stay Awake brand caffeine pills. Figure 1 Figure 2 Acknowledgments I would like to thank Dr. Elizabeth Dolci for the effort she put in to make this experiment possible and the Science Department at Johnson State College for providing the materials and equipment necessary. YPD -TrP -Ile Plates treated with Caffeine as a possible mutagen. 0 1000 2000 3000 4000 5000 6000 7000 0.00% 0.50% 1.00% 1.50% 2.00% 2.50% Number of Colonies Concentration of ems Positive Control (YPD) ems + lawn = 0 colonies 0 1000 2000 3000 4000 5000 6000 7000 0 100 200 300 400 500 Number of Colonies μl of H₂O Negative Control (YPD) H₂O - lawn = 0 colonies 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 0.00% 0.01% 0.02% 0.03% 0.04% 0.05% Number of Colonies Caffeine treatment (YPD) Caffeine Concentration of Caffeine Figure 3 Recommended dosage of one pill dissolved into 475 ml of 0.1 M potassium phosphate buffer. Stock solution of .04% diluted with potassium phosphate buffer, plated, and incubated. .01% .02% .03% 0.4% YPD -TrP -Ile YPD -TrP -Ile YPD -TrP -Ile YPD -TrP -Ile 10 ml of caffeine solution sterilized by filtration. A more detailed example of general methods can be found in Pamela Marshall’s lab procedures,” Using Saccharomyces cerevisiae to Test the Mutagenicity of Household Compounds: An Open Ended Hypothesis-Driven Teaching Lab”. (1) Colonies counted after incubation period
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The Mutagenic Potential of Caffeine on Saccharomyces cerevisiae
Transcript of Mutagenesis Poster
- 1. The Mutagenic Potential of Caffeine on Saccharomyces cerevisiae Greggory A. PerryJohnson State college Department of ScienceABSTRACT METHODS Positive Control (YPD) 7000 Recommended dosage of one Number of Colonies pill dissolved into 475 ml of 10 ml of caffeine solution60005000Caffeine has long been studied as a potential mutagen, and proof has been0.1 M potassium phosphate sterilized by filtration. 4000 provided to support this. The objective of this experiment was to determine buffer. 3000 whether or not the Topcare Stay Awake caffeine pill could have mutagenic2000Caffeine treatment (YPD) properties when introduced into Saccharomyces cerevisiae replication cycle. 1000 ems +9000 Saccharomyces cerevisiae was exposed to 0.01%, 0.02%, 0.03%, and 0.04%Stock solution of .04%8000 Number of Colonies 0concentrations of caffeine and allowed to incubate on YPD, -tryptophan, and 0.00% 0.50%1.00% 1.50% 2.00%2.50%7000diluted with potassium Concentration of ems lawn = 0 colonies 6000 isoleucine agarose plated media to determine mutagenesis activity. Unfortunately,phosphate buffer, plated, 5000 the results were inconclusive. This may have been from the possibility that the4000and incubated. Negative Control (YPD) incorrect strain of yeast was shipped for the experiment. This hypothesis can be 70003000 2000 supported by examining the results of the controls. However, given research done 60001000CaffeineNumber of Colonies on caffeine and its potential to be mutagenic, it would be worth investing in an 50000 0.00% 0.01% 0.02% 0.03% 0.04%0.05% experiment similar to this one.4000 Concentration of Caffeine3000.01%.02%.03% 0.4%2000 HO -1000 0 0 100200 300 400 500Figure 3 YPD YPDYPD YPDl of HOlawn = 0 colonies-TrP-TrP -TrP-TrPINTRODUCTION AND PURPOSE -Ile-Ile -Ile-IleCONCLUSIONSColonies counted after The number of mutated colonies on all plates including the positive controls were insignificant, The D-7 strain of Saccharomyces cerevisiae is used in genetic experiments forincubation period which leads me to believe that the strain of yeast that was used for the experiment was not correct. their tendency to spontaneously mutate, and the fact that yeast are one of the simplest A more detailed example of general methods can be found in Pamela Marshalls lab procedures, This could help to explain why I had no results with the mutagen I chose. I am still curious about my eukaryotic cells. This provides a good opportunity to study compounds because theUsing Saccharomyces cerevisiae to Test the Mutagenicity of Household Compounds: An Open Endedcompound, but further experiments with different strains, and also different concentrations of yeast will tend to mutate if the compound is mutagenic.Hypothesis-Driven Teaching Lab. (1) caffeine are needed to say anything conclusive. Candreva et al. have shown that caffeine at a high enough concentration (3g/l) and The fact that there was growth on YPD plates confirms that the compound is non-toxic at this high enough temperature is responsible for a 5-fold increase in mutation within yeast.RESULTS dosage, which provides a good starting point for future experiments. This is a good example of why The study was based on a product regularly drank in Brazil, Known as Hot Mate controls are needed. Without the controls, it would appear that caffeine had little to no mutagenic Fusion. This common drink was found not to be as mutagenic at room temperature (2). The tryptophan lacking media produced one colony. The isoleucine lacking media produced noproperties. colonies, and one of these plates was infected with mold, as can be seen in figure 2. The product I chose to test was the TopCare Stay Awake pill. This contains 200 Several studies have been conducted on caffeine showing various results, the previously mg of caffeine and small amounts of other compounds, mostly dye. The molecular The number of colonies on the YPD plates confirms that caffeine is not toxic and a good candidate mentioned study conducted by Candreva et al. documented caffeine as a mutagen, but with some structure of caffeine is that of a purine, (See Figure 1below.) which makes up half ofcorrelation to temperature. At 54C the rate of mutation was much higher than at 30C (2). This for this experiment as seen on Figure 3 each base pair in DNA and RNA. This gives caffeine an enormous potential to besuggests high heat is breaking down elements of the compound allowing it to be accepted as a base. mutagenic given the similarities. The positive and negative controls both had significant growth on YPD, while minimal growth occurred on tryptophan and isoleucine lacking media. A study on E. coli showed that caffeine has the potential to inhibit the mechanisms of DNA The concentration I chose to use is much lower than in Hot Mate Fusion, but the repair. Selby and Sancar demonstrated that caffeine impairs DNA repair much in the same way as purpose of this experiment was to determine if the recommended dosage of caffeine ethidium bromide; it inhibits nucleotide excision repair by promoting nonspecific binding of the was mutagenic.Plates treated with Caffeine as a possible mutagen. damage recognition subunit, (3) otherwise it traps the subunit that repairs damage in regions of the DNA that do not need repair. Given the knowledge that has been obtained on caffeine already, I believe it is very important to Molecular formula ofcontinue the research on this potentially harmful compound that is consumed in mass quantities daily suspected mutagen around the world. (caffeine) in TopCare Stay Awake brand caffeine pills. BIBLIOGRAPHY (1) Pamela A. Marshall. Winter 2007. Using Saccharomyces cerevisiae to Test the Mutagenicity of Household Compounds: An Open Ended Hypothesis-Driven Teaching Lab. CBE-Life Sciences Education. Vol:6 pp. 307-315. (2) E. Carmen Candreva, Deborah J. Keszenman, Enrique Barrios, Ulises Gele, and Elia Nunes. 1993. Mutagenicity Induced by Hyperthermia, Hot Mate Infusion, and Hot Caffeine in Saccharomyces cerevisiae. CANCER RESEARCH. Vol:53 pp. 5750-5753. (3) Christopher P. Selby, Aziz Sancar. 1990 Molecular Mechanisms of DNA repair inhibition by caffeine. Proc. Acad. Sci. USA. Vol:87 pp. 3522-3525.Figure 1Acknowledgments I would like to thank Dr. Elizabeth Dolci for the effort she put in to make this experiment possible and the Science YPD-TrP -Ile Figure 2 Department at Johnson State College for providing the materials and equipment necessary.printed bywww.postersession.com
