Mutagen Effects on E. coli Gene Expression

Patrick SkebaPittsburgh Central Catholic

Grade 11

Purpose:

Test the effects of burnt toast exposure on the survivorship and

gene expression of E. coli cells

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

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

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

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

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

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

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

Hypotheses

Part 1• 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

Part 2• 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

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

Burnt Toast Production and Dissolving

1. Placed 4 pieces of room temperature bread on metal tray

2. Preheated oven to 178 degrees Celsius (350 Fahrenheit), then moved tray into the oven

3. Once one side was totally black (5 min), flipped over all of the pieces and repeated bread was totally charred

4. Crushed charred bread into fine powder5. Weighed and added 2 grams of the powder into 18 mL

of SDF, then vortexed into solution6. Removed supernatant, added to sterile conical tube,

then vortexed7. Pour solution through filter paper into a beaker, then

filter again using a syringe and a sterile filter

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

X-gal Procedure0% 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)

0.1 mL 0.1 mL 0.1 mL 0.1 mL

1. Vortexed tubes and allowed them to sit for 15 minutes

2. Pipetted 0.1 mL aliquot onto plates containing x-gal and spread using sterile spreader bars

3. Placed plates, upside down into incubator at 37 degrees Celsius for 24 hours

4. Removed plates and counted colonies

Transformation Procedure1. Calcium-competent DH5-Alpha E. coli tubes were thawed and pooled to serve

as a common cell stock

2. Plasmid/stock/SDF solution was mixed and sat for 10 minutes to allow exposure to the variable

3. Appropriate volume of cells was added into microtubes and mixed4. Each sample was incubated in ice for 45 minutes5. Each sample was heat shocked at 37° Celsius for 5 minutes6. 40 µl from each (6) tube was distributed on LB-amp agar plates (4) and spread7. Plates were incubated for 48 hours8. Colonies on each plate were counted

0% 10% 50%Plasmid (B/7) 5 μL 5 μL 5 μLStock solution 0 μL 1 μL 5μLSDF 5 μL 4 μL 0 μLMicrobe (DH5α)

190 μL 150 μL 150 μL

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

P-value: 0.803

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

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

Limitations and Extensions

Limitations• 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

Extensions• 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

Thank You!