Transcript Skeba mutagen gene expression PJAS

Mutagen Effects on E. coli Gene
Expression
Patrick Skeba
Pittsburgh 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
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Lac Z gene codes for β-galactosidase enzyme in E. coli,
which breaks down the β-gal sugar molecule
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X-gal closely resembles β-gal, so it is also cleaved by
the enzyme; compound produces blue color when
cleaved
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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.
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White colonies = LAC Z disrupted
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Blue Colonies = LAC Z intact
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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
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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
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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 powder
5. Weighed and added 2 grams of the powder into 18
mL of SDF, then vortexed into solution
6. Removed supernatant, added to sterile conical tube,
then vortexed
7. 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 xgal 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 Procedure
0%
1%
10%
50%
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
SDF
0.1 mL
1. Vortexed tubes and allowed them to sit for 15
minutes
2. Pipetted 0.1 mL aliquot onto plates containing xgal 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 Procedure
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Calcium-competent DH5-Alpha E. coli tubes were thawed and pooled to serve
as a common cell stock
0%
10%
50%
Plasmid (B/7)
5 μL
5 μL
5 μL
Stock solution
0 μL
1 μL
5μL
SDF
5 μL
4 μL
0 μL
Microbe
(DH5α)
190 μL
150 μL
150 μL
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
Controls
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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!