Juliana Tambellini Thomas Jefferson High School P HARMACEUTICAL I NFLUENCE ON PROKARYOTIC GENE EXPRESSION

 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.  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 (amp r ), and LAC Z, an intact sequence for alpha-complementary (blue/white screening).

 AMP r – selection marker, indicates which cells successfully incorporated plasmid  LAC Z – simple screen for successful integration of plasmid  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 = AMP r, LAC Z disrupted  Blue Colonies = AMP r 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.

 Investigate possible genetic alterations caused by the common pharmaceutical Advil.  Advil will significantly reduce plasmid transformation efficiency/gene expression.  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

LB-Ampicillin X-gal plates E.Coli Cells on ice Micropipetters Plamid A on ice

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: The cell suspensions were incubated for 30 minutes at room temperature ml was transferred from each tube onto LB-agar plates (6 plates per tube = 18 total) 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

Plate #ControlAdvil Average 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 µl of competent E.coli cells were added to each tube 6.Cells were transformed on ice for 40 minutes 7. The cells were heat shocked for three minutes in a incubator at 37 °C µl of LB media was added to each tube 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 hours 11.Colonies were counted, pictures were taken, and results were analyzed

plasmid sterile water Advil Key Controls (Identical) Advil (0.05 and 0.5) 18 µl sterile water 2 µl plasmid 20 µl total 17 µl sterile water 1 µl Advil 20 µl total 2 µl plasmid 8 µl sterile water 10 µl Advil 2 µl plasmid 20 µl total (4)

Examples of : Control 0.1 Concentration 1.0 Concentration

P = 1.67 x P > 0.05 P < 0.05

 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)

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 C C 13 H 18 O 2 H O

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 Citric Acid C6H8O7C6H8O7 Glycerin C 3 H 5 (OH) 3 Sucrose C 12 H 22 O 11

 Difficult to predict transformation efficiency, thus higher colony counts than desirable  Unable to identify ingredient involved in genetic disruption  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

  ine  Mr. Mark Krotec Teacher - Central Catholic High School Use of lab and equipment Central Catholic High School Carnegie Mellon University Supervisor of Experiment Dr. John Wilson Biostatistician - University of Pittsburgh Advice on Statistics