Do Pharmaceuticals Interfere with Gene

Expression?

Juliana TambelliniThomas 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

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 B-galactosidase 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

Genes1. AMPr – selection marker, indicates which cells successfully incorporated plasmid2. LAC-Z – simple screen for successful integration of a gene into a specific plasmid site

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• 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

SDF2. Components were added

according to the table below:

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

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

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

Conclude: Advil and Sudafed do not interfere with E.coli

survivorship.

P = 0.72

Preparation of Petri Dishes

X-gal plate preparation

1. 900 µl of sterile water and 100 µl of X-gal were mixed in a sterile microtube

2. 100 µl was spread onto each plate

900 µl Sterile water100 µl x-gal

1000 µl

Procedure1. 6 microtubes were arranged in a microtube

rack on ice 2. 2 µl of plasmid were added to each tube3. Amounts of sterile water and medicines were

added to each tube in order to achieve test concentrations

4. 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 minutes7. The cells were heat shocked for three minutes

in a incubator at 37 °C 8. 450 µl of LB was then added to each tube9. 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

hours11. Colonies were counted, pictures were taken,

and results were analyzed

Diagram of Procedure

plasmid

sterile water

Advil

Sudafed

Key

Controls (Identical)

Advil

Sudafed

18 µl sterile water2 µl plasmid

20 µl

17 µl sterile water1 µl Advil

20 µl

2 µl plasmid

8 µl sterile water

10 µl Advil

2 µl plasmid

20 µl

8 µl sterile water

10 µl Sudafed2 µl plasmid

20 µl

17 µl sterile water1 µl Sudafed

2 µl plasmid20 µl

(2)

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

• 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 SchoolUse of lab and equipmentSupervisor of Experiment

• Dr. John Wilson Biostatistician - University of PittsburghAdvice on Statistics