Carbohydrate Influence on UV Stressed Yeast

Jason Beiriger

CCHS, Grade 102nd 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

Glucose

Two structures of D-glucose

• A monosaccharide• Important carbohydrate• Source of energy to

many cells• Stimulates the

production of enzymes which assemble amino acids

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

• Glucose supplementation will not significantly aid the survival of UV stressed Saccharomyces cerevisiae

Alternate Hypothesis

• 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

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 107 cells/mL.

4. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL.

5. 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 Components0% glucose 0.1% glucose 2% glucose

Microbe 0.1 mL 0.1 mL 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 10 mL 10 mL

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.

Procedure1. Liquid exposure

Procedure2. Plated 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. The yeast was suspended using a pipette.8. 0.1 mL aliquots were removed from the tubes and spread onto

YEPD agar plates.9. The plates were exposed to UV radiation in a culture hood for

the following time periods: 0, 40, 100 seconds.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.

ANOVA• Abbreviation for analysis of variance• Statistical test comparing variation within and

between experimental groups

• 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

Anova: Single Factor

SUMMARYGroups Count Sum Average Variance

0% no UV 6 464 77.33333 140.66670% UV 6 189 31.5 91.5

ANOVASource of Variation SS df MS F P-value F crit

Between Groups 6302.083 1 6302.083 54.2893 2.4E-05 4.964603Within Groups 1160.833 10 116.0833

Total 7462.917 11

Glucose Remediation Effects on Cells in Liquid Exposure

0% 5% 10%0

10

20

30

40

50

60

70

80

90

No UV60 sec

7.35E-04

6.02E-07 Significant interac-tion

Concentration of Glucose

Num

ber

of c

olon

ies

2.4E-05

0.057424

1.307E-03

Glucose Remediation Effects on Plated Cells

0.0% 0.1% 2.0%0

10

20

30

40

50

60

70

80

90

No UV

30 sec6.17E-06

8.53E-04

Concentration of Glucose

Num

ber

of c

olon

ies

0.0949

4.45E-02

7.6E-08 Significant interaction

Key QuestionsLiquid 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

• 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

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-violet rays

• Synchronizing the exact times of plating

Further Testing

• 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