1. Mutagen Effects on E. coli Gene Expression
Patrick Skeba
Pittsburgh Central Catholic
Grade 11

2. Purpose:
Test the effects of burnt toast exposure on the survivorship and gene expression of E. coli cells

3. Acrylamide White, odorless, crystalline solid
Prepared on industrial scale for use as thickeners and in dye manufacturing
Discovered in foods, accidentally, by scientists in 2002—potato chips, French fries, and heated bread
2007 study found direct link between foods containing acrylamide and ovarian and womb cancer in women
Currently on the European Chemical Agency’s “substances of very high concern” list

4. Benzopyrene Polycyclic aromatic hydrocarbon compound
Found in coal tar, car exhaust fumes, and all smoke resulting from the combustion of organic material
Determined as the cause of scrotal and skin cancer in chimney sweeps and fuel industry workers in 1933
Listed as a “Group 1 Carcinogen” by the International Agency for Research on Cancer
PAH

5. Why Toast?
Burnt foods, especially meats, potatoes, and wheat, are known to contain trace amounts of benzopyrene and acrylamide
These compounds are produced as a result of incomplete combustion, and are directly related to cancerous tumors in lab rats
Fully toasting bread provides ample charred residue for experimentation, and the chemicals are soluble in water
CEREAL

6. Mutagenesis
Process by which the genetic information of an organism is changed in a stable manner, resulting in a mutation
May occur naturally or by mutagenic agents
DNA modification or damage can lead to cancer in mulicellular organisms
Various mechanisms:
PAHs form reactive oxygen species that form adducts with DNA and block or err replication
Acrylamide may bind to cellular protein, GSH, and change the redox status of the cell, which can directly affect gene expression or various transcription factors

7. Escherichia coli
Escherichia coli (E. coli) – very common, found in intestinal tract of most mammals
There are many strains of E. coli, most are non-pathogenic
Pathogenic strains can cause illness and death in humans
Frequently studied in biology – ubiquitous, simple structure, easily manipulated in the laboratory

8. X-gal Gene Expression Assay
Lac Z gene codes for β-galactosidase enzyme in E. coli, which breaks down the β-gal sugar molecule
X-gal closely resembles β-gal, so it is also cleaved by the enzyme; compound produces blue color when cleaved
X-gal substrate is used to indicate the presence of an intact Lac Z. If Lac Z is intact, β-galactosidase activity is restored, with resulting cleavage of X-gal which leads to characteristic blue colony appearance.
White colonies = LAC Z disrupted
Blue Colonies = LAC Z intact
Cell line used (HB101) is known to contain intact Lac Z

9. Plasmid Expression Assay
Plasmids are naked DNA molecules that are separate from the chromosomal DNA and often circular in shape
Genes included in the plasmid can be expressed by the host cell
The plasmid used for experimentation contains a functional gene for ampicillin resistance, which wild type E. coli cells do not contain
If DNA in plasmid is damaged by the variable, the cells will not survive on ampicillin-infused plates

10. Hypotheses
Part 1
Part 2
Null: cell exposure to charred toast solution will not affect survivorship or number of white colonies
Alternative: exposed cells will have lower survivorship and some colonies will have a white color, indicating a damaged lac-z gene
Null: cells transformed with plasmid exposed to variable will not show difference in survivorship on amp-plates
Alternative: exposed plasmids will have been damaged by the compounds in the solution, and thus the colonies would not grow

11. Materials Bimbo Honey Wheat bread Oven HB101 E. coli strain
DH5-α E. coli strain (calcium competent)
LB Agar plates and broth (1% tryptone, 0.5% yeast, 1% NaCl)
X-gal substrate
“Amp 20” stock solution
Plasmid “B”
Plasmid “7”
Vortex
Centrifuge
Sterile Dilution Fluid (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl)
Micropipettes + Sterile pipettes
1.5 mL micro tubes
20 mL test tubes
20 mL conicle tubes
Ethanol, spreader bars, matches
Gloves, goggles
Test tube racks
Syringe, sterile filter
Beaker
Filter paper
“Klett 60” spectrophotometer

12. Burnt Toast Production and Dissolving
Placed 4 pieces of room temperature bread on metal tray
Preheated oven to 178 degrees Celsius (350 Fahrenheit), then moved tray into the oven
Once one side was totally black (5 min), flipped over all of the pieces and repeated bread was totally charred
Crushed charred bread into fine powder
Weighed and added 2 grams of the powder into 18 mL of SDF, then vortexed into solution
Removed supernatant, added to sterile conical tube, then vortexed
Pour solution through filter paper into a beaker, then filter again using a syringe and a sterile filter

13. Plate Infusion Ampicillin
Create 20% ampicillin stock solution: 0.1 grams of crystalline ampicillin into 5 mL of SDF
Pipette 80 μL into center of LB agar plate
Using sterilized spreader bars, spread aliquot over entire surface
Incubate to allow absorption, then store in refrigerator
Repeat for a total of 24 plates
X-gal
Obtain stock solution of x-gal substrate
Pipette 40 μL aliquot into center
Using sterilized spreader bars, spread aliquot over entire surface
Incubate to allow absorption, then store in refrigerator
Repeat for a total of 20 plates

14. X-gal Procedure Vortexed tubes and allowed them to sit for 15 minutes0% 1%
10%
50%
SDF
9.9 mL
9.8 mL
8.9 mL
4.9 mL
Stock Solution
0 mL
0.1 mL
1 mL
5 mL
Microbe (HB101)
Vortexed tubes and allowed them to sit for 15 minutes
Pipetted 0.1 mL aliquot onto plates containing x-gal and spread using sterile spreader bars
Placed plates, upside down into incubator at 37 degrees Celsius for 24 hours
Removed plates and counted colonies

15. Transformation Procedure
Calcium-competent DH5-Alpha E. coli tubes were thawed and pooled to serve as a common cell stock
Plasmid/stock/SDF solution was mixed and sat for 10 minutes to allow exposure to the variable
Appropriate volume of cells was added into microtubes and mixed
Each sample was incubated in ice for 45 minutes
Each sample was heat shocked at 37° Celsius for 5 minutes
40 µl from each (6) tube was distributed on LB-amp agar plates (4) and spread
Plates were incubated for 48 hours
Colonies on each plate were counted 0%
10%
50%
Plasmid (B/7)
5 μL
Stock solution
0 μL
1 μL
5μL
SDF
4 μL
Microbe (DH5α)
190 μL
150 μL

16. Controls Included to ensure that bacteria and ampicillin were viable
Positive Control: Competent cells administered on LB-agar plates grew a lawn of bacteria
Negative Control: Competent cells, without plasmid, administered on LB-amp plates grew NO bacteria

17. P-value: 0.803

18. Plasmid Exposure Experiment
Experiment was conducted three separate times
First two times, there was no growth on any plate; third trial exhibited hundreds of colonies, but only on the 50x plates
As of yet, no explanation for abnormal growth patterns
Experiment is currently being repeated to acquire definitive results: significant difference in survivorship of cells transformed with exposed plasmid and control will indicate that the variable detrimentally affected the plasmid DNA, preventing proper reproduction and expression

19. Conclusions
First experiment showed no correlation between variable and cell survivorship
However, at the highest tested concentration over 10% of the colonies exhibited a white color
This supports the alternative hypothesis: the charred toast produced a mutagenic effect that prevented the expression of the lac-z gene
Results from second experiment are pending, but based on the first experiment there should be reduced survivorship on amp+ plates.
These results do not prove that burnt toast causes cancer in humans, but they do support the hypothesis that the compounds found in charred foods, such as bread, may lead to DNA damage and mutations

20. Limitations and Extensions
Second experiment was unsuccessful, possibly due to imperfect cells, plasmid, or agar
Charred bread was not autoclaved before being dissolved for the stock solution
Inability to know exact chemical composition of charred bread
Test different brands of bread, or more studied sources of acrylamide such as potato chips and cereal
Conduct second experiment for a fourth (and hopefully final) time under ideal circumstances to gain further data to support the conclusion

21. Thank You!