Juliana TambelliniThomas Jefferson High School

PHARMACEUTICAL INFLUENCE ON

PROKARYOTIC GENE EXPRESSION

E.Coli

Gram negative bacterium that is commonly found in the lower intestine of warm-blooded animals.

E.coli has also been utilized as the most studied prokaryote in biological research.

Competent Cells Cells treated to increase ability

to absorb extraneous DNA, usually plasmids.

Derivative of a much-utilized plasmid known as pGEM 7. Contains a functional sequence for resistance to ampicillin (ampr), and LAC Z, an intact sequence for alpha-complementary (blue/white screening).

Plasmid A

AMPr – selection marker, indicates which cells successfully incorporated plasmid

LAC Z – simple screen for successful integration of plasmid

Genes

X-gal X-gal substrate is used to indicate

the presence of an intact Lac Z. If Lac Z is intact, B-galactosidase activity is restored, with resulting cleavage of X-gal which leads to characteristic blue colony phenotype.

White colonies = AMPr, LAC Z disrupted

Blue Colonies = AMPr and LAC Z intact

Recombinant DNA technology makes use of naturally occurring vectors, or shuttles, of DNA.

Plasmids replicate and contain biological information which is ‘read’ and carried out by the cell.

These plasmids could be introduced to a neighboring bacteria of the same species, possibly conferring some new attribute to that recipient.

Cells which absorb extraneous DNA and express a new characteristic are commonly referred to as transformed cells and the process has been named transformation.

Transformation

Investigate possible genetic alterations caused by the common pharmaceutical Advil.

Purpose

Hypothesis Advil will significantly reduce

plasmid transformation efficiency/gene expression.

Null Hypothesis Advil will not significantly

reduce the transformation efficiency/gene expression.

Calcium-competent DH5α E.coli cells

Plasmid A (pGEM-7)

Liquid Advil

LB agar plates( 1 % tryptone,0.5 % yeast extract, 1% NaCl, 1.5 % agar)

LB-ampicillin agar plates

LB-ampicillin x-gal plates

Microtubes

Sterile water

Large test tubes

Sterile dilution fluid

Ice

Spreader bar

Ethanol

Bunsen Burner

Sterile pipette tips

Micropipettors

Sharpie

Microtube rack

Incubator

Nylon gloves

Materials

LB-Ampicillin X-gal plates

E.Coli Cells on ice

Micropipetters

Plamid A on ice

Photographs of

Materials

Drug toxicity effects on E.coli:

1. 2 test tubes filled with 9.0 ml of SDF

2. Components were added according to the table below:

3. 4. The cell suspensions were incubated for 30 minutes at room temperature

4. 5. 0.1 ml was transferred from each tube onto LB-agar plates (6 plates per tube = 18 total)

5. 6. The plates were incubated for 24 hours at 37 ° C

Preliminary Procedure

Cells Sterile Water Medicine Total

Control 0.1 ml 0.9 ml none 10 ml

Advil 0.1 ml 0.4 ml 0.5 ml 10 ml

Plate # Control Advil

1 99 121

2 105 112

3 114 103

4 98 91

5 89 101

6 104 93

Average 101.5 106.2

Results: Preliminary Procedure

P>.05

Conclude: Advil does not interfere with E.coli survivorship.

1. 12 microtubes were arranged in a microtube rack on ice

2. 2 µl of plasmid were added to each tube 3. Sterile water and medicines were added to

each tube in order to achieve test concentration (diagram)

4. The plasmid was exposed to the medicines for 15 minutes on ice

5. 100 µl of competent E.coli cells were added to each tube

6. Cells were transformed on ice for 40 minutes7. The cells were heat shocked for three minutes

in a incubator at 37 °C 8. 120 µl of LB media was added to each tube9. 100 µl of cells were plated onto the LB-amp-X-

gal plates (2 plates from each microtube)10. The plates were incubated at 37 °C for 48 hours11. Colonies were counted, pictures were taken,

and results were analyzed

Procedure

plasmid

sterile water

Advil

Key

Controls (Identical)

Advil (0.05 and 0.5)

18 µl sterile water2 µl plasmid

20 µl total

17 µl sterile water

1 µl Advil

20 µl total

2 µl plasmid

8 µl sterile water

10 µl Advil2 µl plasmid

20 µl total

(4)

Diagram of Procedure

(4) (4)

Examples of : Control

0.1 Concentration

1.0 Concentration

Results

0 0.0500000000000001 0.50

50

100

150

200

250

300

Advil Effects on Gene Transformation(total growth vs. amount of Advil)

µl of Advil/total µl

Num

ber o

f Col

onie

s

P = 1.67 x 10-8

P > 0.05

P > 0.05

P < 0.05

0 0.1 10

50

100

150

200

250

300

Advil Mutagenic Effects on Lac Z

WhiteBlue

µl of Advil/total µl

Num

ber o

f Col

onie

s

Results

Results

0.0 1.00

0.05

0.1

0.15

0.2

0.25

0

0.09

0

White Colonies Ratio(number of white colonies/number of total

colonies)

µl of Advil/total µ

Num

ber o

f Col

onie

s

0.1

At higher concentrations of Advil the null hypothesis was rejected; the Advil appeared to significantly alter gene expression or transformation

At lower concentrations the null hypothesis was accepted

White colonies (genetic disruption) were observed in the lower concentration (0.1) and not in the higher concentration (1.0)

Conclusion

Children's Advil® Suspension

Active ingredient (in each 5mL): Ibuprofen 100 mg (NSAID)*

Purpose: Fever reducer/Pain reliever

*nonsteroidal anti-inflammatory drug

Reduces fever relieves minor aches and pains due to the common cold, flu, sore throat, headaches and toothaches

Research on Advil

C13H18O2

Children's Advil® Suspension

Inactive ingredients

artificial flavors

Carboxymethycellul-ose sodium

Citric acid

Edetate disodium

Red no. 40

Glycerin

Microcrystaline

Polysorbate 80

Purified water

Sodium benzoate

Sorbitol solution

Sucrose

Xanthan gum

Research on Advil

Citric Acid

C6H8O7

Glycerin

C3H5(OH)3

Sucrose

C12H22O11

Difficult to predict transformation efficiency, thus higher colony counts than desirable

Unable to identify ingredient involved in genetic disruption

Limitations

Extensions Isolate active and inactive ingredients and

perform multiple tests to identify which ingredient causes disruption

Vary exposure time

Sequence DNA of treated plasmid, allowing better interpretation of mutagenic effects

www.usda.gov

http://www.time.com/time/magazine

www.advil.com

References

AcknowledgementsMr. Mark Krotec

Teacher - Central Catholic High SchoolUse of lab and equipment

Central Catholic High SchoolCarnegie Mellon University

Supervisor of Experiment

Dr. John Wilson Biostatistician - University of PittsburghAdvice on Statistics