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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
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