Fracking Chemical Effect on Yeast
Mutagenesis
Cam Coco
Pittsburgh Central Catholic
11th Grade
Problem
How 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 Ingredients
Chemicals
Mg/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
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 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
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
Procedure
1.
2.
3.
4.
5.
6.
Yeast(Saccharomyces cerevisiae) were grown overnight in
sterile YEPD Media.
Samples of the overnight cultures were added to fresh media
in a sterile sidearm flask.
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.
The cultures were diluted in sterile dilution fluid to a
concentration of approximately 10⁵ cells/mL.
The fracking chemicals were sterilized by means of a 0.2
micron syringe filter
The experimental variables were mixed with the appropriate
amounts of SDF to create concentrations of 0%, 0.01%, 0.1%,
1%, and 2%.
Concentration Chart
0%
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 Chart
0%
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
Liquid Pulse
250
Surviving Colonies
P-value:9.763 E-28
200
Liquid Pulse
150
100
50
0
0%
0.01%
0.10%
[Fracking Chemicals]
1%
2%
Fracking Chemical Effect on Yeast
Mutagenesis
(-) Lysine
500
Mutated Colonies
400
P-Value: 4.46E-39
(-) Lysine
300
200
100
0
0%
0.01%
0.10%
[Fracking Chemicals]
1%
2%
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
Dunnett’s Test (-) Lysine
0.01%
t-Value:
2.439
T-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/AppleTreeDes
criptions_M.aspx?page=M
• •http://www.webmd.com/a-to-z-guides/e-coli-infectiontopic-overview
• •http://web.uconn.edu/mcbstaff/graf/Student%20present
ations/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
A-nova(Liquid Pulse)
Anova: Single Factor
SUMMARY
Groups
0
0.0001
0.001
0.01
0.02
Liquid Pulse
Count
8
8
8
8
8
ANOVA
Source of Variation
SS
Between Groups
167506.6
Within Groups
4163
Total
171669.6
Sum
Average
1681 210.125
1489 186.125
1147 143.375
726
90.75
253
31.625
df
Variance
146.6964
79.26786
295.9821
45.07143
27.69643
MS
F
P-value
F crit
4 41876.65 352.0737 9.76E-28 2.641465
35 118.9429
39
A-nova(- Lys)
Anova: Single Factor
SUMMARY
Groups
0
0.0001
0.001
0.01
0.02
(-) Lys
Count
Sum
8
8
8
8
8
ANOVA
Source of Variation
SS
Between Groups
934845.4
Within Groups
5121
Total
939966.4
47
165
965
1897
3338
df
Average
5.875
20.625
120.625
237.125
417.25
Variance
16.98214
13.41071
70.55357
366.125
264.5
MS
F
P-value
F crit
4 233711.4 1597.324 4.46E-39 2.641465
35 146.3143
39
Liquid Pulse Data
0%
210
214
192
195
221
206
216
227
0.01%
194
184
176
191
201
186
175
182
0.10%
124
135
147
128
145
172
164
132
1%
82
97
102
89
91
84
94
87
2%
30
34
32
41
28
27
25
36
(-) Lys Data
0%
6
10
5
0
8
0
7
11
0.01%
21
24
15
27
18
20
19
21
0.10%
112
132
122
109
117
131
125
117
1%
213
226
267
254
215
243
248
231
2%
428
413
392
416
425
399
422
443