Ibuprofen Effect on Prokaryotic Gene ExpressionLuis Espinoza-DelgadoCentral Catholic High SchoolGrade 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 TranscriptionJacob-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 OperonLactose 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 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 anti-inflammatory 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

Generic Dye-Free Infant’s Ibuprofen

Active ingredient: ibuprofen ◦ (recommended dosage: 2mL)

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.

Citric Acid

Sucrose

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 Liquid ibuprofen (C13H18O2) 

36 Conical tubes (15mL) 400mL lambda broth 180 cell culture tubes 400mL Z buffer 80mL ONPG (C12H15NO8)

200mL Na2CO3

1mL lactose (C12H22O11)

15mL glucose (C6H12O6)

Two water baths (37°C-concicals, 28°C-cell culture tubes)

9mL SDS (Sodium Dodecyl Sulfate NaC12H25SO4)

18mL Chloroform (CHCl3)

Spectrophotometer 20

Vortex 0.22 micron syringe filters +

10mL syringe Sharpie Latex gloves Incubator (37°C) Ice bath Sterile pipette tips

Procedure 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

ProcedureFuel 10x (A) X (B) control (0)

(-) carb 1 1 1

(+) lactose (15mins) induction

2 2 2

(+) lactose (15mins),(+) glucose (30mins) repression

3 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 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

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1Ibuprofen Effect on E. coli Gene Expression

Time 30Time 60Time 90Time 120

[Ibuprofen]

Abso

rban

ce (β

-gal

acti

vity

)

con

trol

(-c

arb

)

con

trol

(la

ctos

e)

con

trol

(la

ctos

e+gl

uco

se)

x (

-car

b) x (

lact

ose)

x (

lact

ose

+gl

uco

se)

10x

(-c

arb

)

10x

(la

ctos

e)

10x

(la

ctos

e+gl

uco

se)

control x 10x0

0.01

0.02

0.03

0.04

0.05

0.06

Ibuprofen Effect on E.coli Gene Induction at Time 30

(-carb)(lactose)(lactose+glucose)

[Ibuprofen]

Abso

rban

ce (β

-gal

acti

vity

)

P-value=0.2311264

P-value= 0.2673580

P-value= 0.203958

control x 10x0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1Ibuprofen Effect on E.coli Gene Repression at Time 120

(-carb)(lactose)(lactose+glucose)

[Ibuprofen]

Abso

rban

ce (β

-gal

acti

vity

)

P-value= 8.66E-05

P-value= 4.19E-09

P-value=3.42E-06

ConclusionsThe 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.

Unable to identify ingredient involved in possible genetic disruption

Presence of sucrose in liquid ibuprofen as inactive ingredient

Vary pharmaceutical exposure times

Explore different mechanisms for testing gene expression (e.g., x-gal)

Test different modelsUse different

pharmaceutical drugs and higher concentrations

Limitations Extensions

References 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 SS df MS F P-value F crit

Between Groups 11.98535 5 2.397069 5.851149 0.001137 2.620654

Within Groups 9.8322 24 0.409675

Total 21.81755 29        

F>Fcrit-null rejected