Carbohydrate Influence on UV Stressed Yeast Jason Beiriger CCHS, Grade 10 2nd 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 can lead to compounding stress Results in cellular degeneration May cause direct cell death or induce cancer High glucose levels induce an increase in enzymatic activity leading to the synthesis of amino acids

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

Two structures of D-glucose A monosaccharide Important carbohydrate Source of energy to many cells Stimulates the production of enzymes which assemble amino acids Two structures of D-glucose Could supplementing the cell with carbohydrates influence their stress protein response?

Yeast Easy to grow and culture Unicellular Most studied cell in the world Saccharomyces cerevisiae Similar cell cycle, biochemistry and genetics to other eukaryotic cells, like those in humans

Objective/Purpose To determine if carbohydrate supplementation will be effective in protecting Saccharomyces cerevisiae from UV light stress

Null Hypothesis Alternate Hypothesis Glucose supplementation will not significantly aid the survival of UV stressed Saccharomyces cerevisiae Glucose 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, 0.1mM 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 Glucose Incubator YEPD media

Procedure Saccharomyces cerevisiae was grown overnight in sterile dilution YEPD media. A sample of the overnight culture was added to fresh media in a sterile sidearm flask. 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 107 cells/mL. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL. The glucose was diluted with sterile dilution fluid to the chosen concentrations to a total of 9.9 mL. For example: 1 mL of 20% glucose solution + 8.9 mL of SDF = final concentration of almost 2% glucose. (the addition of 0.1 mL of cell culture will result in a total of 10 mL and a 2% concentration)

Chart of Test Tube Components 0% glucose 0.1% glucose 2% glucose Microbe 0.1 mL SDF 9.9 mL 9.4 mL 8.9 mL Glucose stock (2g/10mL) 0 mL 0.5 mL 1 mL Total 10 mL

Procedure 1. Liquid exposure 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 103 cells/mL. 7. 1 mL of the solution was transferred into each of 6 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. 0.1 mL aliquots were removed from the tubes and spread onto YEPD agar plates. 10. The plates were incubated at 30 degrees Celsius for 48 hours. 11. The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

Procedure 2. Plated Exposure 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 103 cells/mL. The yeast was suspended using a pipette. 0.1 mL aliquots were removed from the tubes and spread onto YEPD agar plates. The plates were exposed to UV radiation in a culture hood for the following time periods: 0, 40, 100 seconds. The plates were incubated at 30 degrees Celsius for 48 hours. The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

Sample ANOVA used in experiment Abbreviation for analysis of variance Statistical test comparing variation within and between experimental groups Anova: Single Factor SUMMARY Groups Count Sum Average Variance 0% no UV 6 464 77.33333 140.6667 0% UV 189 31.5 91.5 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 6302.083 1 54.2893 2.4E-05 4.964603 Within Groups 1160.833 10 116.0833 Total 7462.917 11   If the P- value is lower than the alpha value (.05), then the result is significant (a result of the variable influence) Sample ANOVA used in experiment

Glucose Remediation Effects on Cells in Liquid Exposure 0.057424 1.307E-03 Number of colonies 2.4E-05 Concentration of Glucose

Glucose Remediation Effects on Plated Cells 0.0949 7.6E-08 Significant interaction 4.45E-02 Number of colonies Concentration of Glucose

Key Questions Liquid Exposure Plated Exposure Did UV hurt the cells? 2.4E-05 Significant Did sugar hurt the cells? 0.057424 Insignificant Did 5% sugar hurt the cells? 0.28007 Insignificant Did 10% sugar hurt the cells? 0.135139 Insignificant Did sugar help remediate UV damage? 6.02E-07 Significant interaction Plated Exposure Did UV hurt the cells? 6.17E-06 Significant Did sugar hurt the cells? 0.0949 Insignificant Did 5% sugar hurt the cells? 4.45E-03 Significant Did 10% sugar hurt the cells? 0.085 Insignificant Did sugar help remediate UV damage? 7.6E-08 Significant interaction

Conclusion Null Hypothesis Hypothesis Carbohydrate supplementation will not significantly aid the survival of UV stressed Saccharomyces cerevisiae REJECTED by analysis Carbohydrate supplementation will significantly aid the survival of UV stressed Saccharomyces cerevisiae SUPPORTED by analysis

Limitations Further Testing 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-violet rays Synchronizing the exact times of plating More replicates Utilize various wavelengths of UV light Different concentrations of glucose Different carbohydrate

Sources www.FDA.com http://www.ncbi.nlm.nih.gov/pubmed/8097593 http://bioinfo.hku.hk/services/analyseq/cgi-bin/proteol_in.pl Fraser-Reid, Bert, "van't Hoff's Glucose", Chem. Eng. News 77 (39): 8 Fred W. Schenck “Glucose and Glucose-Containing Syrups” in Ullmann's Encyclopedia of Industrial Chemistry 2006, Wiley-VCH, Weinheim. doi: 10.1002/14356007.a12_457.pub2 Solubility of D-glucose in non-aqueous solvents, http://oru.edu/cccda/sl/solubility/allsolvents.php?solute=D-glucose