Transcript pharmaceutical effects on gene expresson Edited Tambellini

Do Pharmaceuticals
Interfere with Gene
Expression?
Juliana Tambellini
Thomas Jefferson High School
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.
Plasmid A
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).
(2977 base pairs)
LAC Z
AMP r
Genes
1. AMPr – selection marker, indicates which cells
successfully incorporated plasmid
2. LAC-Z – simple screen for successful
integration of a gene into a specific plasmid site
X-gal
• In gene cloning, the X-gal substrate is used to
indicate the presence of an intact Lac Z. If Lac Z
is intact, alpha complementation restores Bgalactosidase activity, with resulting cleavage of
X-Gal which leads to characteristic blue colony
phenotype.
• This technique allows for the quick and easy
detection of successful gene integration into
plasmid, without the need to individually test
each colony.
• White colonies = AMPr, LAC Z disrupted
• Blue colonies = AMPr , and LAC-z intact
Transformation
• Recombinant DNA technology often 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.
• Natural Plasmids can be introduced to a
neighboring bacteria of the same species,
possibly conferring some new attribute to
that recipient. (antibiotic resistance)
Research on Pharmaceuticals
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
Non-Drowsy Children’s Sudafed ®
• Active Ingredient (in each 5mL): Pseudoephedrine
HCL 15 mg
– Purpose: Nasal Decongestant
• Temporarily reduces congestion due to common cold or
other respiratory allergies, promotes nasal and/or sinus
draining
• Required to show identification
when purchasing
Purpose
• Investigate possible genetic alterations
caused by common pharmaceuticals.
Hypothesis
• The pharmaceuticals will significantly
reduce plasmid transformation
efficiency/gene expression.
Null Hypothesis
• The pharmaceuticals will not significantly
reduce plasmid transformation efficiency/
gene expression.
Materials
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Calcium-competent DH5α E.coli cells
Plasmid A (pGEM-7)
X-gal
Liquid Advil
Liquid Sudafed
LB agar plates( 1 % tryptone,0.5 % yeast extract,
1% NaCl, 1.5 % agar)
LB-ampicillin agar 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
Preliminary Procedures
Drug toxicity effects on E.coli:
1. 3 test tubes filled with 9.0 ml of SDF
2. Components were added according to
the table below:
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
Sudafed
0.1 ml
0.4 ml
0.5 ml
10 ml
4.. The cell suspensions were incubated
for 30 minutes at room temperature
5. 0.1 ml aliquots were transferred from
each tube onto LB-agar plates (6
plates per tube = 18 total)
6. The plates were incubated for 24 hours
at 37 ° C
Results: Preliminary Procedure
Plate #
Control
Advil
Sudafed
1
99
121
116
2
105
112
125
3
114
103
99
4
98
91
107
5
89
101
89
6
104
93
101
Average
101.5
103.5
106.2
P = 0.72
Conclude: Advil and Sudafed do not
interfere with E.coli survivorship.
Preparation of Petri Dishes
X-gal plate preparation
1. 900 µl of sterile water and 100 µl of Xgal were mixed in a sterile microtube
2. 100 µl was spread onto each plate
900 µl Sterile water
100 µl x-gal
1000 µl
Procedure
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6 microtubes were arranged in a microtube rack on
ice
2 µl of plasmid were added to each tube
Amounts of sterile water and medicines were added
to each tube in order to achieve test concentrations
The plasmid was exposed to the medicines for 30
min on ice
5. 100 µl of competent E.coli cells were added to each
tube
6. Cells were incubated on ice for 40 minutes
7. The cells were heat shocked for three minutes in a
incubator at 37 °C
8. 450 µl of LB was then added to each tube
9. 100 µl of cells were plated onto LB-amp-X-gal plates
(5 plates from each microtube)
10. The plates were incubated at 37 °C for 48 hours
11. Colonies were counted, pictures were taken, and
results were analyzed
Diagram of Procedure
Controls (Identical)
18 µl sterile water
2 µl plasmid
(2)
20 µl
Advil
17 µl sterile water
8 µl sterile water
1 µl Advil
10 µl Advil
2 µl plasmid
2 µl plasmid
20 µl
20 µl
Sudafed
8 µl sterile water
17 µl sterile water
1 µl Sudafed
10 µl Sudafed
2 µl plasmid
2 µl plasmid
20 µl
20 µl
Key
plasmid
Advil
sterile water
Sudafed
Conclusion
•At higher concentrations of
pharmaceuticals the null hypothesis was
rejected; the agents appeared to
significantly alter gene expression or
transformation
• At lower concentrations of
pharmaceuticals the null was accepted
•Advil appeared to have a greater
negative impact on transformation/gene
expression than Sudafed
Limitations
• Difficult to predict transformation
efficiency, thus higher colony counts than
desirable
• Different inactive ingredients in each
pharmaceutical
• X-gal plating technique may have lead to
errors in blue/white screening
Extentions
• Perform a preliminary experiment in
which multiple amounts of transformed
E.coli cells are plated to gauge the
number of colonies produced
• Focus on one medication and use
varying concentrations
•Vary pharmaceutical exposure times
• Infuse dishes with x-gal to ensure
blue/white screening accuracy
Sources
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www.pharmacy.org
http://www.hopkinsmedicine.org/Research/
www.usda.gov
http://www.time.com/time/magazine
www.advil.com
www.sudafed.com
Acknowledgements
• Mr. Mark Krotec
Teacher - Central Catholic High School
Use of lab and equipment
Supervisor of Experiment
• Dr. John Wilson
Biostatistician - University of Pittsburgh
Advice on Statistics