Palm Oil Effects On Yeast Mutagenesis Peter Koltas Central Catholic High School 3rd Year in Pjas 11th grade

Palm Oil An edible vegetable oil derived from the mesocarp (reddish pulp) of the fruit of the oil palm Many processed foods either contain palm oil or various ingredients derived from it European Food Safety Authority said more tests are needed to determine if it is carcinogenic or if it is safe to eat

Question Is commercially sold Palm Oil a potential mutagen?

Cell Model Saccharomyces cerevisiae Commonly used model Tolerant and safe to culture Has similar reproduction,
metabolism, and chemistry
as other more advanced eukaryotic cells (-) Lys Special strain unable to produce lysine

Lysine Lysine codons AAA, AAG (-) lysine yeast mutants used in research Lys 2 mutants are missing an enzyme function within the lysine biosynthesis pathway Result: Cells require lysine supplementation

Ames Test Used a (-)-histidine mutant Salmonella
(single-point substitution)
Bacteria cannot synthesize histidine due to mutation Exposure to suspected mutagen correlated with increased reversion (mutation) rate A lower limit on mutation, assayed only 1 DNA site in genome

Modified Ames Test 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+)

Purpose To determine the effects of Palm oil (suspected mutagen) on the mutagenesis rate of (-) Lys yeast.

Hypotheses Null Hypothesis: Palm oil will not have a significant effect on the mutagenesis rate of yeast. Alternate Hypothesis: Palm oil will have a significant effect on the mutagenesis rate of yeast.

Materials Vortex Spreader bar Ethanol Micro burner (-) Lysine agar plates 1% yeast nitrogen base w/o amino acids 2% glucose 1 mM amino acid mix 1.5% agar Sterile dilution fluid (SDF)
10 mM KH2PO4, 1 mM MgSO4, 1 mM CaCl2, 100 mM NaCl SDF Test Tubes Macro/micropipettes Sterile pipette tips, microplates Vortex Spreader bar Ethanol Micro burner (-) Lysine Saccharomyces cerevisiae Rubber gloves Test tubes Microtubes Test tube rack Palm Oil Incubator

Procedure Strain of yeast (-) Lys phenotype grown for 2 days in YEPD media. Sterile yeast pellet washed with SDF to remove any residual nutrients (lysine) and allowed to sit and acclimate Stock re-suspended and stored in com. (-) Lys media for 2 days A 10% sub-stock of the Palm oil was made by diluting the variable with sterile water. Sterilized through a 0.22 micron syringe filter The pellet was re-suspended in SDF

Procedure (cont.) The following ingredients were pipetted into sterile 1.5 mL microtubules: Sterile Water Yeast Palm Oil Total volume 0% 1.04 ml 0.26 ml 0 ml 1.3 ml 0.1% substock 1.03 ml 0.013ml 1% 0.013 ml 10% 0.91 ml 0.13 ml

Procedure (cont.) 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) 12 plates for each concentration, totaling 48 plates 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

Results P value= 0.02 control

ANOVA Statistical test that allows for the comparison of means of different groups, to determine significant variation Single factor ANOVA Utilizes p-values as measure of significance P>0.05: not significant P<0.05: significant

Dunnett’s Test Used to find out which variable groups produced significant variation compared to a control If T-value is greater than the T critical, variations are considered significant

Dunnett’s Test Results T critical= 2.353 Test T Value Interpretation 0% vs. 0.1% 0.98 Not Significant 0% vs. 1% 2.73 Significant 0% vs. 10% 3.81

Interpretations of Results The 1% and 10% concentrations of Palm oil showed the ability to significantly affect the rate of yeast mutagenesis Null hypothesis can be rejected Alternate hypothesis can be accepted

Limitations and Inconsistencies Slightly out-of-synced plating which leads to slightly different exposure times to palm oil Inability to control the exact amount of cells on each plate (minor difference overshadow by massive amount of cells) Slight positioning differences during the incubation process Surprisingly low number of revertants

Extensions Reduction of lab time with more assistants Trypan Blue Assay to account for dead cells A future experiment testing palm oil’s effects on cancerous cell lines to see if it promotes uncontrollable growth Repeat the experiment with a higher concentration of cells.

Bibliography http://www.saynotopalmoil.com/Whats_the_issue.php http://wwf.panda.org/what_we_do/footprint/agriculture/palm_oil/ http://metro.co.uk/2017/01/12/what-is-palm-oil-and-can-it-give-you-cancer-after-the-nutella-scare-6376936/ http://www.biology-pages.info/A/AmesTest.html