typs - 2012.igem.org
TRANSCRIPT
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TYPS Tübingen yeast progesterone screen iGEM 2012 Tübingen University
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Our Team
06/10/2012 Team Tübingen 2012 2
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Intersex fish
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„The prevalence of intersex is more widespread than anyone anticipated“ Haseltine, U.S.G.S.
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What are intersex fish?
Male fish are concerned
Less fertile
Developement of ovotestis
→ Endocrine disruptors
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Water Contamination
Medical waste
Fertilizers, intensive farming
Chemical industries
Unknown factors
→ Seawage treatment?
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Endocrine disruptors
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? BPA
Progesterone 17β-Estradiol
Biologically active in low concentrations
Synergistic effects
Difficult to detect, Bioassays
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1.Receptor 2.Inverter 3.Reporter
TYPS (Tübingen yeast progesterone screen)
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The receptor: mPRα
Extracellular
Specific
Heterologous expression in yeast
Source organisms: a) Danio rerio
b) Xenopus laevis
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a)
b)
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The receptor: mPRα
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Padh1 mPR Tadh1
Pfet3
Jessica L. Smith et. al: “Heterologous expression of human mPRα, mPRβ and mPRγ in yeast confirms their ability to function as membrane progesterone receptors” Steroids, 2008
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The promoter: Pfet3
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Pfet3
Active in low-iron environment
LIM (low-iron medium)
Repressed by iron receptors of the PAQR superfamily
→ Negative signal
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The inverter
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Pfet3 Repressor
PRepressor Reporter
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The inverter
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Pfet3 Repressor
PRepressor Reporter
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The inverter
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Pfet3 Repressor
PRepressor Reporter
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The inverter
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A) Mig1 / Psuc2 Glucose-dependent gene regulation
B) Rox1 / Panb1 Oxygen-dependent gene regulation
Well characterized repression systems
Knockout strains available
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The reporter
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A) ß-Galactosidase Several substrates available
CPRG, high absorbtion
Used in YES-assays
CPRG
B) Firefly-Luciferase Bioluminescence
High sensitivity
Signal sequence deleted, no transport to peroxisomes
Luciferine
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TYPS
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(Tübingen yeast progesterone screen)
Plasmids: pRS313 pRS315 pRS316
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Flexibility
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Lab impressions
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Project status
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Promoters Genes Terminator
Padh1
Pfet3 mPRα (X.laevis)
Mig1
mPRα (D.rerio) Tadh1
Panb1
Psuc2
ROX1
Luciferase
LacZ
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Human practice
06/10/2012 Team Tübingen 2012 20
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Team Tübingen thanks…
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Advisors: Prof. Ralf-Peter Jansen and lab
Prof. Alfred Nordheim Prof. Lutz Heide
Prof. Oliver Kohlbacher Dr. Christian Apel
Sponsors
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Measuring endocrine disruptors
Currently available assays
Vitellogenin test (in vivo)
Human breast cancer cells
YES (yeast estrogen screen)-assays
→ TYPS (Tübingen yeast progesterone screen)
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Measurement
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Read out
SPE of water samples
Reference substrances
TYPS assay culture
Incubation & Lysis