1. Fracking Chemical Effect on Yeast Mutagenesis Cam Coco Pittsburgh Central Catholic 11 th Grade

2. Problem How do fracking chemicals influence the mutagenesis of Saccharomyces cerevisiae?

3. Fracking Background Fracking is used to access natural gas from shale that is deep underground Hydraulic Fracturing is effective, however it may be unhealthy for the environment. Many nature groups have spoken against the practice of hydraulic fracturing. The root of the problem is the chemical solution used in mining the gas. Are living organisms at risk from fracking?

4. Fracking Chemical Ingredients ChemicalsMg/l Sodium Chloride 75.650 Calcium Chloride 16.650 Magnesium Chloride 0.4750 Barium Chloride 0.1851 Krypton Chloride 0.3846 Strontium Chloride 0.200 Due to strict regulations, couldn’t obtain actual fluids, a researcher at CMU provided a simulation formula mix which was created along with the help of a chemistry instructor

5. Yeast Common cell model Tolerant and safe to culture Has similar reproduction, metabolism, and chemistry as other more advanced eukaryotic cells Saccharomyces cerevisiae Special strain that is unable to produce Lysine was used – Lys 2 (-)

6. Lysine’s codons are AAA and AAG In research here are defined minus lysine yeast mutants used Lys 2 mutants are missing an enzyme function within the lysine biosynthesis Result – cells require lysine supplement

8. Ames Test Developed to test the mutagenic and anti-mutagenic properties of various chemicals by Bruce Ames in 1970s Ames used minus histidine mutant Salmonella (single point substitution) Exposure to suspected mutagen correlated with increased reversion (mutation) rate Visible colonies appearing on complete (-His) media give evidence of mutation through reversion Only 1 DNA site in genome assayed

9. Modified Ames Test (-)Lys Yeast–Eukaryote The number of reverted colonies of yeast can be correlated with the rate of mutation A reversion at that point can result in a reversion back to wild type yeast (lys +) Anti-Mutagen – Exposure to UV Mutagen(Fracking Chemicals) – No exposure needed

10. Purpose To determine whether fracking chemicals have a significant effect on the mutagenesis of Saccharomyces cerevisiae

11. Hypotheses Null Hypothesis: The fracking chemicals will not have a significant effect on the mutagenesis of Yeast Alternative Hypothesis: The fracking chemicals will have a significant effect on the mutagenesis Saccharomyces cerevisiae.

12. Materials Fracking Chemicals Micro and Macro pipettes + Sterile tips Spreader bars Saccharomyces Cerevisiae (obtained from Wolford Lab) Complete (-) Lysine agar plates Complete (-) Lysine agar Burner Sidearm Flask Vortex Incubator Gloves/goggles SDF (Sterile Dilution Fluid) 20 Sterile Test Tubes Ethanol YEPD Agar Plates(To be Infused with Agar Plates) 0.22 micron syringe filters and 10mL syringe Klett Spectrophotometer

13. Procedure 1.Yeast(Saccharomyces cerevisiae) were grown overnight in sterile YEPD Media. 2.Samples of the overnight cultures were added to fresh media in a sterile sidearm flask. 3.The cultures were placed in an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 7 cells/mL. 4.The cultures were diluted in sterile dilution fluid to a concentration of approximately 10⁵ cells/mL. 5.The fracking chemicals were sterilized by means of a 0.2 micron syringe filter 6.The experimental variables were mixed with the appropriate amounts of SDF to create concentrations of 0%, 0.01%, 0.1%, 1%, and 2%.

14. Concentration Chart 0%0.01%0.1%1%2% Sterile Fluid added 1mL0.999mL0.99mL0.9mL0.8ml Yeast0.1mL.1ml Fracking Chemicals 0mL0.001mL0.01mL0.1mL.2ml Original Sterile Fluid 8.9mL 8.9ml Total Volume 10mL 10ml

15. Procedure 7.100 µL of cell culture was then added to the fracking chemical solutions, yielding a final volume of 10 mL and a cell density of approximately 10 3 cells/mL. 8.The solutions were vortexed and allowed to sit at room temperature for 15 minutes. 9.After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on YEPD-agar plates. 10.The plates were incubated at 30°C for 48 hours. 11.The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

16. (-) Lysine Procedure A strain of yeast (-) Lys phenotype was grown for 2 days in complete (-) Lysine media The cells were washed with complete (-) Lysine media and aloud to sit to acclimate Fracking chemicals where sterilized through a 0.22 micron syringe filters The yeast pellet was re-suspended in SDF The following ingredients were pipetted into sterile micro tubes. (Percent are by volume compared to stock solution)

17. Concentration Chart 0%0.01%0.1%1%2% Fracking Chemicals [10%] 0mL0.001mL0.01mL0.1ml0.2mL Sterile Dilution Fluid 0.8mL0.799mL0.79mL0.7mL0.6mL Yeast0.2mL Total Volume 1mL

18. (-) Lysine Procedure The cells were allowed to sit for 15 minutes 0.2 mL aliquots were spread onto complete (-) Lys agar plates (necessary to show cells that have reverted through mutation to wild type (+) Lys) All plates were allowed to incubate for 5 days at 32 ̊C The colonies were counted and recorded. Each colony assumed to have arisen from 1 cell

19. Fracking Chemical Effect on Yeast Survivorship P-value:9.763 E-28 [Fracking Chemicals] Surviving Colonies

20. Fracking Chemical Effect on Yeast Mutagenesis P-Value: 4.46E-39 Mutated Colonies [Fracking Chemicals]

21. Dunnett's Test (Survivorship) 0.01%t-Value: 4.401 T-Crit: 3.02 Significant 0.1%t-Value: 12.241 T-Crit: 3.02 Significant 1%t-Value: 21.891 T-Crit: 3.02 Significant 2%t-Value: 31.734 T-Crit: 3.02 Significant

22. Dunnett’s Test (-) Lysine 0.01%t-Value: 2.439 T-Crit: 3.02Insignificant 0.1%t-Value: 18.973 T-Crit: 3.02Significant 1%t-Value: 38.236 T-Crit: 3.02Significant 2%t-Value: 68.018 T-Crit: 3.02Significant

23. Conclusions Reject null hypothesis for all concentrations except 0.01% The increase in the concentration of fracking chemicals is generally correlated with the increase of mutagenesis of the Yeast

24. Limitations Plating could have been slightly unsynchronized Only 5 concentrations were tested Only one exposure time

25. Future Studies Use more concentrations of fracking chemicals Vary exposure times Use various cell models Trypan blue assay

26. Works Cited http://www.maplevalleyorchards.com/Pages/AppleTreeDes criptions_M.aspx?page=M http://www.webmd.com/a-to-z-guides/e-coli-infection- topic-overview http://web.uconn.edu/mcbstaff/graf/Student%20presenta tions/S%20epidermidis/sepidermidis.html http://www.life.umd.edu/classroom/bsci424/BSCI223Web SiteFiles/GramPosvsGramNeg.htm http://www.specialtyproduce.com/produce/McIntosh_Ap ples_1228.php

27. A-nova(Liquid Pulse)

28. A-nova(- Lys)

29. Liquid Pulse Data

30. (-) Lys Data