TYPS T bingen y east p rogesterone s creen iGEM 2012 Tbingen - PowerPoint PPT Presentation

TYPS T übingen y east p rogesterone s creen iGEM 2012 Tübingen University

Our Team 06/10/2012 Team Tübingen 2012 2

Intersex fish „The prevalence of intersex is more widespread than anyone anticipated “ Haseltine, U.S.G.S. 06/10/2012 Team Tübingen 2012 3

What are intersex fish?  Male fish are concerned  Less fertile  Developement of ovotestis → Endocrine disruptors 06/10/2012 Team Tübingen 2012 4

Water Contamination  Medical waste  Fertilizers, intensive farming  Chemical industries  Unknown factors → Seawage treatment? 06/10/2012 Team Tübingen 2012 5

Endocrine disruptors ?  Biologically active in low BPA concentrations  Synergistic effects  Difficult to detect, Bioassays Progesterone 17 β -Estradiol 06/10/2012 Team Tübingen 2012 6

(Tübingen yeast TYPS progesterone screen) 1.Receptor 2.Inverter 3.Reporter 06/10/2012 7

The receptor: mPR α  Extracellular b)  Specific  Heterologous a) expression in yeast  Source organisms: a) Danio rerio b) Xenopus laevis 06/10/2012 8

The receptor: mPR α Padh1 mPR Tadh1 Jessica L. Smith et. al: “Heterologous expression of human mPR α, mPR β and mPR γ in yeast confirms their ability to function as Pfet3 membrane progesterone receptors” Steroids, 2008 06/10/2012 9

The promoter: Pfet3  Active in low-iron environment  LIM (low-iron medium)  Repressed by iron Pfet3 receptors of the PAQR superfamily → Negative signal 06/10/2012 10

The inverter P Repressor Reporter Pfet3 Repressor 06/10/2012 11

The inverter P Repressor Reporter Pfet3 Repressor 06/10/2012 12

The inverter P Repressor Reporter Pfet3 Repressor 06/10/2012 13

The inverter A) Mig1 / Psuc2 B) Rox1 / Panb1 Glucose-dependent gene Oxygen-dependent gene regulation regulation  Well characterized repression systems  Knockout strains available 06/10/2012 14

The reporter A) ß-Galactosidase  Several substrates available Luciferine  CPRG, high absorbtion  Used in YES-assays B) Firefly-Luciferase  Bioluminescence  High sensitivity  Signal sequence deleted, no transport to peroxisomes CPRG 06/10/2012 15

(Tübingen yeast TYPS progesterone screen) Plasmids: pRS313 pRS315 pRS316 06/10/2012 Team Tübingen 2012 16

Flexibility 06/10/2012 Team Tübingen 2012 17

Lab impressions 06/10/2012 Team Tübingen 2012 18

Project status Promoters Genes Terminator mPR α ( D.rerio ) Tadh1 Padh1 Pfet3 mPR α ( X.laevis ) Psuc2 Mig1 Panb1 ROX1 Luciferase LacZ 06/10/2012 Team Tübingen 2012 19

Human practice 06/10/2012 Team Tübingen 2012 20

Team Tübingen thanks …  Advisors: Prof. Ralf-Peter Jansen and lab Prof. Alfred Nordheim Prof. Lutz Heide Prof. Oliver Kohlbacher Dr. Christian Apel  Sponsors 06/10/2012 Team Tübingen 2012 21

Measuring endocrine disruptors Currently available assays  Vitellogenin test ( in vivo )  Human breast cancer cells  YES (yeast estrogen screen)-assays → TYPS (Tübingen yeast progesterone screen) 06/10/2012 Team Tübingen 2012 22

Measurement Reference substrances SPE of water samples Read out Incubation & Lysis TYPS assay culture 06/10/2012 Team Tübingen 2012 23