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.