Fracking Chemical Effect on Yeast Mutagenesis

Cam CocoPittsburgh Central Catholic

11th Grade

ProblemHow do fracking chemicals influence the mutagenesis of Saccharomyces cerevisiae?

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?

Fracking Chemical IngredientsChemicals Mg/l

Sodium Chloride

75.650

Calcium Chloride

16.650

Magnesium Chloride

0.4750

Barium Chloride

0.1851

Krypton Chloride

0.3846

StrontiumChloride

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

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 (-)

• 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

Ames Test

• Developed to test the mutagenic andanti-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

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

Purpose

• To determine whether fracking chemicals have a significant effect on the mutagenesis of Saccharomyces cerevisiae

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.

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

Procedure1. 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 107 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%.

Concentration Chart0% 0.01% 0.1% 1% 2%

Sterile Fluid added

1mL 0.999mL 0.99mL 0.9mL 0.8ml

Yeast 0.1mL 0.1mL 0.1mL 0.1mL .1ml

Fracking Chemicals

0mL 0.001mL 0.01mL 0.1mL .2ml

Original Sterile Fluid

8.9mL 8.9mL 8.9mL 8.9mL 8.9ml

Total Volume

10mL 10mL 10mL 10mL 10ml

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 103 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.

(-) 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)

Concentration Chart0% 0.01% 0.1% 1% 2%

Fracking Chemicals [10%]

0mL 0.001mL 0.01mL 0.1ml 0.2mL

Sterile Dilution Fluid

0.8mL 0.799mL 0.79mL 0.7mL 0.6mL

Yeast 0.2mL 0.2mL 0.2mL 0.2mL 0.2mL

Total Volume

1mL 1mL 1mL 1mL 1mL

(-) 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

Fracking Chemical Effect on Yeast Survivorship

0.00% 0.01% 0.10% 1.00% 2.00%0

50

100

150

200

250

Liquid Pulse

Liquid Pulse

P-value:9.763 E-28

[Fracking Chemicals]

Surviving Colonies

Fracking Chemical Effect on Yeast Mutagenesis

0.00% 0.01% 0.10% 1.00% 2.00%0

50100150200250300350400450

(-) Lysine

(-) Lysine

P-Value: 4.46E-39 Mutated Colonies

[Fracking Chemicals]

Dunnett's Test (Survivorship)0.01% t-Value:

4.401T-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

Dunnett’s Test (-) Lysine0.01% t-Value:

2.439T-Crit: 3.02 Insignificant

0.1% t-Value:18.973

T-Crit: 3.02 Significant

1% t-Value:38.236

T-Crit: 3.02 Significant

2% t-Value:68.018

T-Crit: 3.02 Significant

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

Limitations

• Plating could have been slightly unsynchronized

• Only 5 concentrations were tested

• Only one exposure time

Future Studies

• Use more concentrations of fracking chemicals

• Vary exposure times

• Use various cell models

• Trypan blue assay

Works Cited

• http://www.maplevalleyorchards.com/Pages/AppleTreeDescriptions_M.aspx?page=M

• •http://www.webmd.com/a-to-z-guides/e-coli-infection-topic-overview

• •http://web.uconn.edu/mcbstaff/graf/Student%20presentations/S%20epidermidis/sepidermidis.html

• •http://www.life.umd.edu/classroom/bsci424/BSCI223WebSiteFiles/GramPosvsGramNeg.htm

• •http://www.specialtyproduce.com/produce/McIntosh_Apples_1228.php

A-nova(Liquid Pulse)Anova: Single Factor Liquid Pulse

SUMMARYGroups Count Sum Average Variance

0 8 1681 210.125 146.69640.0001 8 1489 186.125 79.26786

0.001 8 1147 143.375 295.98210.01 8 726 90.75 45.071430.02 8 253 31.625 27.69643

ANOVASource of Variation SS df MS F P-value F critBetween Groups 167506.6 4 41876.65 352.0737 9.76E-28 2.641465Within Groups 4163 35 118.9429

Total 171669.6 39

A-nova(- Lys)Anova: Single Factor (-) Lys

SUMMARYGroups Count Sum Average Variance

0 8 47 5.875 16.982140.0001 8 165 20.625 13.41071

0.001 8 965 120.625 70.553570.01 8 1897 237.125 366.1250.02 8 3338 417.25 264.5

ANOVASource of Variation SS df MS F P-value F critBetween Groups 934845.4 4 233711.4 1597.324 4.46E-39 2.641465Within Groups 5121 35 146.3143

Total 939966.4 39

Liquid Pulse Data0% 0.01% 0.10% 1% 2%

210 194 124 82 30214 184 135 97 34192 176 147 102 32195 191 128 89 41221 201 145 91 28206 186 172 84 27216 175 164 94 25227 182 132 87 36

(-) Lys Data0% 0.01% 0.10% 1% 2%

6 21 112 213 42810 24 132 226 413

5 15 122 267 3920 27 109 254 4168 18 117 215 4250 20 131 243 3997 19 125 248 422

11 21 117 231 443