Team:Tuebingen/ProjectImplementation

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{{:Team:Tuebingen/Template/Tuebingen}}
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= Implementation =
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__TOC__
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The following plasmid constructs are needed to implement the measurement system. Using two receptors, two signalling systems and two reporter genes the implementation contains redundancy to select for a sensitive system.
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<!--
 +
<table class="wikitable">
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<tr><th>backbone pRS#</th><th>part #1</th><th>part #2</th><th>part #3</th></tr>
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<tr><td>313</td><td>Padh1</td><td>mPR ''Danio rerio''</td><td>Tadh1</td></tr>
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<tr><td>313</td><td>Padh1</td><td>mPR ''Xenopus laevis''</td><td>Tadh1</td></tr>
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<tr><td>315</td><td>Pfet3</td><td>rox1</td><td>Tadh1</td></tr>
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<tr><td>315</td><td>Pfet3</td><td>mig1</td><td>Tadh1</td></tr>
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<tr><td>316</td><td>Panb1</td><td>lacZ</td><td>Tadh1</td></tr>
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<tr><td>316</td><td>Panb1</td><td>luciferase</td><td>Tadh1</td></tr>
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<tr><td>316</td><td>Psuc2</td><td>lacZ</td><td>Tadh1</td></tr>
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<tr><td>316</td><td>Psuc2</td><td>luciferase</td><td>Tadh1</td></tr>
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</table>
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-->
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== Plasmid 1 ==
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'''Task:''' Expression of membrane receptor.
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{| class="wikitable"
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|-
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! backbone pRS# !! part #1 !! part #2 !! part #3
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|-
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| 313 || Padh1 || mPR ''Danio rerio'' || Tadh1
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|-
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| colspan=4 | [[File:PRS313_Padh1_Danio_Tadh1.png|membrane construct #1]]
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|-
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| 313 || Padh1 || mPR ''Xenopus laevis'' || Tadh1
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|-
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| colspan=4 | [[File:PRS313_Padh1_Xenopus_Tadh1.png|membrane construct #2]]
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|}
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== Implementation ==
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== Plasmid 2 ==
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'''Task:''' Signalling, inhibitor controlled by membrane receptor through Pfet3.
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{| class="wikitable"
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|-
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! backbone pRS# !! part #1 !! part #2 !! part #3
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|-
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| 315 || Pfet3 || rox1 || Tadh1
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|-
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| colspan=4 | ''targets Plasmid 3 with Panb1''<br/> [[File:PRS315_Pfet3_rox1_Tadh1_V3.png|inhibitor construct #1]]
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|-
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| 315 || Pfet3 || mig1 || Tadh1
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|-
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| colspan=4 | ''targets Plasmid 3 with Psuc2''<br/> [[File:pRS315_Pfet3_mig1_Tadh1.png|inhibitor construct #2]]
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|}
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== Plasmid 3 ==
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=== Promoter ===
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'''Task:''' Expression of reporter gene, controlled by inhibitor (plasmid 2).
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[[File:PRS313_Padh1_Danio_Tadh1.png|promoter1]]
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{| class="wikitable"
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[[File:PRS313_Padh1_Xenopus_Tadh1.png|promoter2]]
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|-
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=== Inhibitor ===
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! backbone pRS# !! part #1 !! part #2 !! part #3
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[[File:PRS315_Pfet3_rox1_Tadh1_V3.png|inhibitor1]]
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|-
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[[File:pRS315_Pfet3_mig1_Tadh1.png|inhibitor2]]
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| 316 || Panb1 || luciferase || Tadh1
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=== Reporter ===
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|-
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[[File:PRS316_Panb1_lacZ_Tadh1.png|reporter1]]
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| colspan=4 | [[File:PRS316 Psuc2 Luciferase Tadh1.png|reporter construct #1]]
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[[File:pRS316_Psuc2_lacZ_Tadh1.png|reporter2]]
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|-
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[[File:PRS316_Psuc2_Luciferase_Tadh1.png|reporter3]]
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| 316 || Panb1 || lacZ || Tadh1
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|-
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| colspan=4 | [[File:PRS316_Panb1_lacZ_Tadh1.png|reporter construct #2]]
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|-
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| 316 || Psuc2 || luciferase || Tadh1
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|-
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| colspan=4 | [[File:PRS316_Psuc2_Luciferase_Tadh1.png|reporter construct #3]]
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|-
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| 316 || Psuc2 || lacZ || Tadh1
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|-
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| colspan=4 | [[File:pRS316_Psuc2_lacZ_Tadh1.png|reporter construct #4]]
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|}
== Measurement ==
== Measurement ==
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because these produce signals which can simply be quantified by optical
because these produce signals which can simply be quantified by optical
measurement methods.
measurement methods.
-
 
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== References ==
 
-
 
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* [1] Jessica L. Smith, Brian R. Kupchak, Ibon Garitaonandia, L. Kim Hoang, Andrew S. Maina, Lisa M. Regalla, and Thomas J. Lyons - 2008 - Heterologous expression of human mPRα, mPRβ and mPRγ in yeast confirms their ability to function as membrane progesterone receptors
 
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* [2] Sumpter, Johnson (2008) Reflections on endocrine disruption in the aquatic environment
 
-
<!--
 
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* Liew et. al. - 2012 Polygenic Sex Determination System in Zebrafish
 
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* Hanna et al. - 2006 - Cell-surface expression, progestin binding, and
 
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rapid nongenomic signaling of zebrafish membrane progestin receptors
 
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alpha and beta in transfected cells
 
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* Reupke - 2011 - Detektion dopingrelevanter anaboler Steroide in
 
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Pferdeurin und Pferdeplasma mithilfe eines Reportergen-Assays in
 
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Hefezellen. (Detection of doping relevant anabolic steroids in horse
 
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urin and blood plasma using yeast reporter gene assays)
 
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* “Assault on the male”, BBC 1996. Video report
 
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http://www.youtube.com/watch?v=LkxIJJI37bQ
 
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* Dr. Volker Scheil, The impact of potential environmental stressors on
 
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early development and cellular and biochemical biomarkers in fish,
 
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Main research: Fish embryotoxicity, histopathology and stress protein
 
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(hsp 70) responses.
 
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* Harris et al. - 2011 - The consequences of feminization in breeding
 
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groups of wild fish
 
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* Liew et al. - 2012 - Polygenic Sex Determination System in Zebrafish
 
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* Sumpter, Johnson - 2008 - 10th Anniversary Perspective Reflections on
 
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endocrine disruption in the aquatic environment from known knowns to
 
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unknown unknowns (and many things in between)
 
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* Werner, Palace, Wautier - 2006 - Reproductive fitness of lake trout
 
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(Salvelinus namaycush) exposed to environmentally relevant
 
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concentrations of the potent estrogen ethynylestradiol (EE2) in a
 
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whole lake exposure experiment
 
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* Fenske, M., Maack, G., Schäfers, C., & Segner, H. (2005). An
 
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environmentally relevant concentration of estrogen induces arrest of
 
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male gonad development in zebrafish, Danio rerio. Environmental
 
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toxicology and chemistry / SETAC, 24(5), 1088-98. Retrieved from
 
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http://www.ncbi.nlm.nih.gov/pubmed/16110986 (kein PDF)
 
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* Schäfers, C., Teigeler, M., Wenzel, A., Maack, G., Fenske, M., &
 
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Segner, H. (2007). Concentration- and time-dependent effects of the
 
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synthetic estrogen, 17alpha-ethinylestradiol, on reproductive
 
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capabilities of the zebrafish, Danio rerio. Journal of toxicology and
 
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environmental health. Part A, 70(9), 768-79.
 
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doi:10.1080/15287390701236470
 
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* Segner, H., Caroll, K., Fenske, M., Janssen, C. R., Maack, G., Pascoe,
 
-
D., Schäfers, C., et al. (2003). Identification of
 
-
endocrine-disrupting effects in aquatic vertebrates and invertebrates:
 
-
report from the European IDEA project. Ecotoxicology and environmental
 
-
safety, 54(3), 302-14. Retrieved from
 
-
http://www.ncbi.nlm.nih.gov/pubmed/12651186 (kein PDF)
 
-
* Hanna et al. - 2006 - Cell-surface expression, progestin binding, and
 
-
rapid nongenomic signaling of zebrafish membrane progestin receptors
 
-
alpha and beta in transfected cells
 
-
* Thomas et al. - 2007 - Steroid and G protein binding characteristics
 
-
of the seatrout and human progestin membrane receptor alpha subtypes
 
-
and their evolutionary origins
 
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* Thomas - 2008 - Characteristics of membrane progestin receptor alpha
 
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(mPR ) and progesterone membrane receptor component 1 (PGMRC1) and
 
-
their roles in mediating rapid progestin actions
 
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* Jessica L. Smith, Brian R. Kupchak, Ibon Garitaonandia, L. Kim Hoang,
 
-
Andrew S. Maina, Lisa M. Regalla, and Thomas J. *Lyons  - 2008 -
 
-
Heterologous expression of human mPRα, mPRβ and mPRγ in yeast confirms
 
-
their ability to function as membrane progesterone receptors
 
-
 
-
-->
 

Latest revision as of 09:27, 24 September 2012



Implementation

Contents

The following plasmid constructs are needed to implement the measurement system. Using two receptors, two signalling systems and two reporter genes the implementation contains redundancy to select for a sensitive system.


Plasmid 1

Task: Expression of membrane receptor.

backbone pRS# part #1 part #2 part #3
313 Padh1 mPR Danio rerio Tadh1
membrane construct #1
313 Padh1 mPR Xenopus laevis Tadh1
membrane construct #2

Plasmid 2

Task: Signalling, inhibitor controlled by membrane receptor through Pfet3.

backbone pRS# part #1 part #2 part #3
315 Pfet3 rox1 Tadh1
targets Plasmid 3 with Panb1
inhibitor construct #1
315 Pfet3 mig1 Tadh1
targets Plasmid 3 with Psuc2
inhibitor construct #2

Plasmid 3

Task: Expression of reporter gene, controlled by inhibitor (plasmid 2).

backbone pRS# part #1 part #2 part #3
316 Panb1 luciferase Tadh1
reporter construct #1
316 Panb1 lacZ Tadh1
reporter construct #2
316 Psuc2 luciferase Tadh1
reporter construct #3
316 Psuc2 lacZ Tadh1
reporter construct #4

Measurement

The measurement itself should be limited to an optical one. With this idea in mind, we decided for reporter genes like lacZ and luciferase, because these produce signals which can simply be quantified by optical measurement methods.

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