1. Do Pharmaceuticals Interfere with Gene Expression? Juliana Tambellini Thomas Jefferson High School

2. 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.

3. Plasmid A Derivative of a much-utilized plasmid known as pGEM 7. Contains a functional sequence for resistance to ampicillin (amp r ), and LAC Z, an intact sequence for alpha-complementary (blue/white screening). (2977 base pairs) LAC Z AMP r

4. 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 = AMP r, LAC Z disrupted Blue colonies = AMP r, and LAC-z intact Genes 1. AMP r – selection marker, indicates which cells successfully incorporated plasmid 2. LAC-Z – simple screen for successful integration of a gene into a specific plasmid site

5. 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)

6. 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

7. 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.

8. 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

9. 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: 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 CellsSterile WaterMedicineTotal Control0.1 ml0.9 mlnone10 ml Advil0.1 ml0.4 ml0.5 ml10 ml Sudafed0.1 ml0.4 ml0.5 ml10 ml

10. Results: Preliminary Procedure Plate #ControlAdvilSudafed 199121116 2105112125 311410399 49891107 58910189 610493101 Average101.5103.5106.2 Conclude: Advil and Sudafed do not interfere with E.coli survivorship. P = 0.72

11. 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 µ l Sterile water 100 µ l x-gal 1000 µ l

12. Procedure 1.6 microtubes were arranged in a microtube rack on ice 2.2 µ l of plasmid were added to each tube 3.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 µ l 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 µ l of LB was then added to each tube 9.100 µ l 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

13. Diagram of Procedure plasmid sterile water Advil Sudafed Key Controls (Identical ) Advil Sudafed 18 µ l sterile water 2 µ l plasmid 20 µ l 17 µ l sterile water 1 µ l Advil 20 µ l 2 µ l plasmid 8 µ l sterile water 10 µ l Advil 2 µ l plasmid 20 µ l 8 µ l sterile water 10 µ l Sudafed 2 µ l plasmid 20 µ l 17 µ l sterile water 1 µ l Sudafed 2 µ l plasmid 20 µ l (2)

14. 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

15. 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

16. 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 School Use of lab and equipment Supervisor of Experiment Dr. John Wilson Biostatistician - University of Pittsburgh Advice on Statistics