amino acid remediation of uv stressed yeast jason beiriger cchs, grade 9 1st year in pjas
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Amino Acid Remediation of UV Stressed Yeast
Jason Beiriger
CCHS, Grade 91st Year in PJAS
Ultraviolet Rays
• Light waves that have shorter wavelengths, thus greater energy, than visible light
• They range from 400nm to 10nm
• Given off from the sun but most are absorbed by the ozone layer
Damage due to UV light
• Damage includes skin burn, sun poisoning, skin irritation, redness, photo-aging, nausea, and possibly skin cancer
• FDA Protection methods include sun screen, hats, and radiation-blocking clothing
• Can cause DNA to form dimers, leading to replication errors, mutations
Oxidative stress
• UV light can also result in oxidation stress• Increases oxidant production in cells• Free Radical accumulation compounding
stress• Results in cellular degeneration • Could cause direct cell death or induce cancer• Antioxidants are thought to counter oxidative
stress
Antioxidants• Antioxidant- a molecule
capable of preventing the oxidation of other molecules
• Oxidation- a chemical reaction that transfers electrons from a substance to an oxidizing agent
• Oxidation reactions can produce free radicals, which can damage cells
Stress Proteins
• Free Radicals can disrupt the shape and function of many molecules of life such as lipids, carbohydrates, proteins, and sometimes even nucleic acids
• Stress proteins are involved in restoring the structure and function of critical cell proteins that have been damaged by stress
Proteins• Proteins are polymers of
amino acids• Could supplementing
the cell with amino acids aid their stress protein response?
• Peptone is a peptic digest of a population of bacterial proteins (small peptides + free amino acids)
• Peptone is commonly used as a source of amino acids in microbial growth media
Yeast • Most studied cell in the world
• Easy to grow and culture
• Similar cell cycle, biochemistry and genetics to other eukaryotic cells, like those in human skin
• Saccharomyces cerevisiae
Problem
• UV light radiation is harmful and is able to kill cells
Objective/Purpose
• To determine if amino acid supplementation will be effective in protecting Saccharomyces cerevisiae from UV light stress
Null Hypothesis
• Peptone supplementation will not significantly aid the survival of UV stressed Saccharomyces cerevisiae
Hypothesis
• Peptone supplementation will significantly aid the survival of UV stressed Saccharomyces cerevisiae
Materials
• 60 YEPD agar plates(1% yeast extract, 2% peptone, 2% dextrose, 1.5% agar)• Sterile dilution fluid [SDF] (10mM KH2PO4, 10mM K2HPO4, 1mM MgSO4, .1mM CaCl2, 100mM
NaCl)• Klett spectrophotometer• Sterile pipette tips and Micropipettors• Vortex• Sidearm flask• Spreader bar• Ethanol• Micro burner• Saccharomyces cerevisiae (yeast)• UV Hood• Rubber Gloves• Test tubes• Test Tube Rack• SDF Test Tubes• Microtubes• Peptone• Incubator• YEPD media
Procedure
1. Saccharomyces cerevisiae was grown overnight in sterile dilution YEPD media.2. A sample of the overnight culture was added to fresh media in a sterile
sidearm flask.3. The culture was incubated at 30 degrees Celsius until a density of 50 Klett
spectrophotometer units was reached. This represents a cell density of approximately 10ˆ7 cells/ml.
4. The culture was diluted in sterile dilution fluid to a concentration of approximately 10ˆ5 cells/ml.
5. The peptone was diluted with sterile dilution fluid to the chosen concentrations to a total of 9.9 ml. For example:
1 ml. of 10% peptone solution + 8.9 ml. of SDF = final concentration of almost 1% peptone. (the addition of 0.1 ml. of cell culture will result in a total of 10 ml. and a 1% concentration)
6. 0.1 ml. of cell culture was then added to the test tubes, yielding a final volume of 10 ml. and a cell density of approximately 10ˆ3 cells/ml.
7. 1 ml of the solution was transferred into each of 18 microtubes. The microtubes were exposed to UV radiation in a culture hood for the following time periods. 0, 40, 100 seconds
8. After UV exposure, the yeast was suspended using a pipette.9. The solution was mixed by vortexing and allowed to sit at room
temperature for 15 minutes.10. After vortexing to evenly suspend cells, 0.1 ml. aliquots were
removed from the tubes and spread onto YEPD agar plates.11. The plates were incubated at 30 degrees Celsius for 48 hours.12. The resulting colonies were counted. Each colony is assumed to
have arisen from one cell.
Anova• Abreviation for analysis of variance• Statistical test to see variance between and
within groups
• If the P- value is larger than the alpha value (.05), then the result is significant
Sample ANOVA used in experiment
Amino Acid Remediation of UV Stressed Yeast
0 40 1000
20
40
60
80
100
120
140
0% peptone
0.1% peptone
1% peptone
Exposure Time (seconds)
P= .20665
P= 4.87E-13 P= 3.42E-06
# of
sur
vivi
ng c
olon
ies
Dunnet’s TestVariable Comparison T value
compared tot critical value
Result
40 second UV exposure to control 6.55>2.86 significant
100 second UV exposure to control 13.45>2.86 significant
40 second UV exposure and .1% amino acid to control (.1% amino acid)
7.1>2.86 significant
40 second UV exposure and .1% amino acid to control (.1% amino acid)
7.3>2.86 significant
40 second UV exposure and 1% amino acid to control (1% amino acid)
1.9<2.86 insignificant
100 second UV exposure and 1% amino acid to control (1% amino acid)
1.9<2.86 insignificant
Results- Key Questions• Did amino acid concentrations significantly affect the survival of yeast
stressed by UV radiation?– Interaction P-value 2.58E-10 Significant
• Did the amino acid concentration affect cell survivorship without UV exposure?– P-value .20665 Insignificant
• Did UV exposure affect cell survivorship?– P-value 4.36E-09 Significant
• Did amino acid affect cell survivorship at 40 seconds of UV exposure?– P-value 3.42E-06 Significant
• Did amino acid affect cell survivorship at 100 seconds of UV exposure?– P-value 4.87E-13 Significant
Conclusion
• Peptone supplementation will not significantly aid the survival of UV stressed Saccharomyces cerevisiae
• NOT SUPPORTED by data
• Peptone supplementation will significantly aid the survival of UV stressed Saccharomyces cerevisiae
• SUPPORTED by data
Null Hypothesis Hypothesis
Limitations
• Due to slight differences in positioning in the UV hood, the cultures may have received slight differences in the amount of exposure to the ultra-violent rays.
• Synchronizing the exact times of plating.
Further Testing
• More replicates• Utilize various
wavelengths of UV light.• Perform SDS gel
electrophoresis on the yeast protein population searching for characteristic stress protein responses
Sources• www.FDA.com • http://www.ncbi.nlm.nih.gov/pubmed/8097593• http://bioinfo.hku.hk/services/analyseq/cgi-bin/proteol_in.pl• Payne JW (1976). "Peptides and micro-organisms". Advances in
Microbial Physiology 13: 55–113. doi:10.1016/S0065-2911(08)60038-7. PMID 775944.http://www.phys.ksu.edu/
• Finking R, Marahiel MA (2004). "Biosynthesis of nonribosomal peptides1". Annual Review of Microbiology 58: 453–88. doi:10.1146/annurev.micro.58.030603.123615. PMID 15487945.
• Duquesne S, Destoumieux-Garzón D, Peduzzi J, Rebuffat S (August 2007). "Microcins, gene-encoded antibacterial peptides from enterobacteria". Natural Product Reports 24 (4): 708–34. doi:10.1039/b516237h. PMID 17653356.
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