Ibuprofen Effect on Prokaryotic
Gene Expression
Luis Espinoza-Delgado
Central Catholic High School
Grade 11
β-galactosidase
Gene Expression
• Cells in different parts of the same organism
•
•
differ in both their structure and physiology,
despite containing identical DNA.
Cell development- the selective turning on
and off of genes with various chemical
messengers.
Gene expression is used by all known life to
generate macromolecules specific to the
functions and needs of a given cell.
o
Products are often proteins.
Gene Transcription

Jacob-Monod model of prokaryotic gene
induction
◦ Production of enzyme depends on the presence of an
inducer (transcription factor).
◦ Structural genes specify the amino acid sequence of
the enzyme.
◦ Regulator gene controls the transcription of the
structural genes by synthesizing a repressor protein.

Operon- DNA sequence of the promoter,
operator, and immediate structural genes
◦ RNA polymerase- promoter
◦ Repressor protein- operator
Lac Operon
Lactose inactivates a repressor protein, allowing
RNA polymerase to transcribe structural genes
β-galactosidase
 ONPG- similar structure which is broken down
into galactose and ortho-nitrophenol (yellow)

No lactose present
Repressor releases when lactose is
present
Escherichia coli
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Escherichia coli is a large and diverse
group of gram (-) bacteria.
It is found in the intestinal tract of
most animals, including humans
Most strains of E. coli are harmless,
others cause sickness.
Estimated to cause infection in more
than 70,000 patients a year in the
United States.
Reported to cause both large
outbreaks as well as isolated
sporadic infections in small numbers
of individuals.
Serves as a common prokaryotic cell
model.
Ibuprofen
NSAID used to treat arthritis, primary dysmenorrheal, and
fever; also serves as an analgesic.
 Inhibits cyclooxygenase- produces prostaglandins that
promote inflammation, pain, and fever.
 Non selective of the isoforms of cyclooxygenase it inhibits.
◦ Inhibition of COX-2 enzyme leads to the antiinflammatory properties
◦ COX-1 inhibition affects platelet aggregation and the
gastrointestinal tract
◦ Side effects of this drug include: upset stomach,
mild heartburn, diarrhea, constipation; bloating, gas;
dizziness, headache, nervousness; chest pain, weakness of
heart, slurred speech; rapid weight gain; nausea; fever;
bruising, muscle weakness; and sensitivity to light.

Pharmaceutical Research
Citric Acid
Generic Dye-Free Infant’s Ibuprofen
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Active ingredient: ibuprofen
◦ (recommended dosage: 2mL)
Sucrose
Purpose: Fever reducer/Pain reliever
Reduces fever relieves minor aches and pains due to the
common cold, flu, sore throat, headaches and
toothaches.
• Inactive ingredients: anhydrous citric acid,
butyleparaben, flavor, glycerin, hypromellose,
polysorbate 80, propylene glycol, purified
water, sodium benzoate, sorbitol solution,
sucrose, and xanthan gum.
Does ibuprofen affect microbial
flora in humans?
Purpose:
To assess the effect(s) of ibuprofen on
the gene expression of E. coli
Null Hypothesis:
Ibuprofen will not have a significant effect
on the gene expression of E. coli.
Alternative Hypothesis:
Ibuprofen will have a significant effect on
the gene expression of E. coli.
Materials

Escherichia coli
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Vortex
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Liquid ibuprofen (C13H18O2)
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36 Conical tubes (15mL)
0.22 micron syringe filters + 10mL
syringe
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400mL lambda broth

Sharpie
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180 cell culture tubes

Latex gloves

400mL Z buffer
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Incubator (37°C)

80mL ONPG (C12H15NO8)
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Ice bath
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200mL Na2CO3
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Sterile pipette tips
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1mL lactose (C12H22O11)
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15mL glucose (C6H12O6)
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Two water baths (37°C-concicals,
28°C-cell culture tubes)
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9mL SDS (Sodium Dodecyl Sulfate
NaC12H25SO4)
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18mL Chloroform (CHCl3)
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Spectrophotometer 20
Procedure
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E. coli was grown overnight in sterile lambda media.
A sample of the overnight culture was added to fresh media in a
sterile sidearm flask.
The culture was placed in a shaking water bath (37°C) until a
density of 50 Klett spectrophotometer units was achieved. This
represents a cell density of approximately 107 cells/mL.
50mL of lambda broth were extracted from a flask containing
400mL and replaced with 50mL of E. coli. The resulting mixture was
swirled and allowed to sit for 5 minutes.
The selected experimental variables were diluted with the lambda/
E. coli mixture.
10x
x
0x
Model/lambda 9mL
9.9mL
10mL
Ibuprofen
1mL
0.1mL
0mL
Total volume
10mL
10mL
10mL
Procedure
Fuel
10x (A)
X (B)
control (0)
(-) carb
1
1
1
(+) lactose (15mins)
induction
2
2
2
(+) lactose (15mins), 3
(+) glucose (30mins)
repression
3
3
• Added 2.0mL ice-cold Z buffer to each culture tube.
•Removed 200µL aliquots from all conical tubes. Placed aliquots into their
designated time 0 culture tubes in the ice cold bath.
•Placed conical tubes into 37°C water bath and removed every 5 minutes to
invert 10 times.
•After 15 minutes, added 100µL of lactose to conical tubes A2, B2,02,A3, B3,
and 03. After an additional 15 minutes, added 1mL of glucose to conical tubes
A3, B3, and 03.
•Removed 200µL aliquots from conical tubes and added to the appropriate
tubes at the indicated time. Returned culture tubes to water bath and inverted
every 5 minutes. Repeated this step every 30 minutes until time 120.
Procedure
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Added 50µL aliquots of SDS into each culture tube, followed by
100µL of chloroform. Tubes were mixed thoroughly with vortex.
Placed test tube rack into top water bath (28°C). Allowed
samples to sit for 5 minutes.
Added 400µL aliquots of ONPG into each culture tube.
Mixed tubes by hand and then returned to water bath.
After 15 minutes, stopped enzyme reaction by adding 1.0mL of
Na2CO3 to each culture tube.
Took spectrophotometer reading to measure absorbance of each
sample with the wavelength set to 420nm.
Performed the appropriate statistical analyses to adequately assess
the data.
0.2
0.1
0
0.4
0.3
[Ibuprofen]
10x (lactose)
10x (lactose+glucose)
10x (-carb)
x (lactose +glucose)
x (lactose)
x (-carb)
control (lactose+glucose)
control (lactose)
control (-carb)
Absorbance (β-gal activity)
Ibuprofen Effect on E. coli Gene Expression
1
0.9
0.8
0.7
0.6
Time 30
0.5
Time 60
Time 90
Time 120
Ibuprofen Effect on E.coli Gene Induction at Time 30
0.06
P-value= 0.2673580
Absorbance (β-gal activity)
0.05
P-value=0.2311264
P-value= 0.203958
0.04
0.03
(-carb)
(lactose)
(lactose+glucose)
0.02
0.01
0
control
x
[Ibuprofen]
10x
Ibuprofen Effect on E.coli Gene Repression at Time 120
1
P-value= 4.19E-09
Absorbance (β-gal activity)
0.9
0.8
P-value=3.42E-06
P-value= 8.66E-05
0.7
0.6
0.5
(-carb)
0.4
(lactose)
(lactose+glucose)
0.3
0.2
0.1
0
control
x
[Ibuprofen]
10x
Conclusions
The null hypothesis can be accepted for
all time intervals.
 Ibuprofen did not significantly alter the
ability of E. coli to turn its lac operon on
and off.
 Different energy mechanisms clearly
affected gene expression.
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Limitations
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Unable to identify
ingredient involved in
possible genetic
disruption
Presence of sucrose in
liquid ibuprofen as
inactive ingredient
Extensions
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Vary pharmaceutical
exposure times
Explore different
mechanisms for
testing gene
expression (e.g., x-gal)
Test different models
Use different
pharmaceutical drugs
and higher
concentrations
References
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http://www.americanheart.org
http://bio.classes.ucsc.edu
http://www.bio.cmu.edu/lacOperon
http://www.britannica.com/genetic-expression
http://www.hopkinsmedicine.org/research
http://www.medicinenet.com
http://www.mun.ca/biochem
http://www.ncbi.nlm.nih.gov
http://oregonstate.edu/regulation
Freeman, Scott. Biological Science. San Fransicso, CA: Benjamin
Cummings, 2011.
“Turning Genes On and Off in Bacteria” Dr. Linda Roman
Kauffman, Department of Biological Sciences at Carnegie
Mellon University
ANOVA
• Abbreviation for analysis of variance
• Statistical test to see variance between and
within groups
• If the F value is higher than the F crit, then
there is significant variation in the data.
• P value lower the alpha cutoff gives a high
degree of confidence in this interpretation.
• Such case warrants the use of Dunnett’s test.
ANOVA Statistical Analysis
Source of Variation
Between Groups
Within Groups
Total
SS
df
MS
11.98535
5
2.397069
9.8322
24
0.409675
21.81755
29
F
P-value
5.851149
0.001137
F>Fcrit-null rejected
F crit
2.620654