SLIDE 1
TYPS
Tübingen yeast progesterone screen
iGEM 2012 Tübingen University
Our Team
06/10/2012 Team Tübingen 2012 2
TYPS T bingen y east p rogesterone s creen iGEM 2012 Tbingen - - PowerPoint PPT Presentation
TYPS T bingen y east p rogesterone s creen iGEM 2012 Tbingen - - PowerPoint PPT Presentation
TYPS T bingen y east p rogesterone s creen iGEM 2012 Tbingen University Our Team 06/10/2012 Team Tbingen 2012 2 Intersex fish The prevalence of intersex is more widespread than anyone anticipated Haseltine, U.S.G.S. 06/10/2012
SLIDE 2
SLIDE 3
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
SLIDE 7
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1.Receptor 2.Inverter 3.Reporter
TYPS
(Tübingen yeast progesterone screen)
SLIDE 8
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
SLIDE 10
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
SLIDE 11
The inverter
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Pfet3 Repressor PRepressor Reporter
SLIDE 12
The inverter
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Pfet3 Repressor PRepressor Reporter
SLIDE 13
The inverter
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Pfet3 Repressor PRepressor Reporter
SLIDE 14
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
SLIDE 15
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
SLIDE 16
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
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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
SLIDE 22
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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SLIDE 23
Measurement
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Read
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SPE of water samples Reference substrances TYPS assay culture Incubation & Lysis