TYPS Tübingen yeast progesterone screen iGEM 2012 Tübingen University

Our Team

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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.

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)

The receptor: mPRα

Extracellular

Specific

Heterologous expression in yeast

Source organisms: a) Danio rerio

b) Xenopus laevis

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a)

b)

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

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

The inverter

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Pfet3 Repressor

PRepressor Reporter

The inverter

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Pfet3 Repressor

PRepressor Reporter

The inverter

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Pfet3 Repressor

PRepressor Reporter

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

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

TYPS

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(Tübingen yeast progesterone screen)

Plasmids: pRS313 pRS315 pRS316

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

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

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