Team:Tuebingen/ProjectImplementation

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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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 +
<!--
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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>
 +
<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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! 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]]
 +
|}
 +
 +
== 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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! backbone pRS# !! part #1 !! part #2 !! part #3
 +
|-
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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 ==
 +
'''Task:''' Expression of reporter gene, controlled by inhibitor (plasmid 2).
 +
{| class="wikitable"
 +
|-
 +
! backbone pRS# !! part #1 !! part #2 !! part #3
 +
|-
 +
| 316 || Panb1 || luciferase || Tadh1
 +
|-
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| colspan=4 | [[File:PRS316 Psuc2 Luciferase Tadh1.png|reporter construct #1]]
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|-
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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 ==
 +
 +
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.

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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