* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download pharmaceutical effects on gene expresson Edited Tambellini
Survey
Document related concepts
Cell culture wikipedia , lookup
Gene expression profiling wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Gene regulatory network wikipedia , lookup
Cell-penetrating peptide wikipedia , lookup
Genetic engineering wikipedia , lookup
Endogenous retrovirus wikipedia , lookup
List of types of proteins wikipedia , lookup
Gene therapy of the human retina wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
DNA vaccination wikipedia , lookup
Transcript
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 • • • • • • • • • • • • • • • • • • • • • 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 1. 2. 3. 4. 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 • • • • • • 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