Team:INSA Toulouse/contenu/lab practice/results/red sensor

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   <h1 class="title1">Results</h1>
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   <h1 class="title1">Results - Red Light Sensor Characterization</h1>
    
    
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   <h2 class="title2">Red Light Sensor Characterization</h2>
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   <h2 class="title2">Objective</h2>
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<p class="texte">Characterize the red light sensor system using Cph8 with its promoter pOmpc. Reminder: in darkness, the EnvZ histidine kinase of Cph8 phosphorylates endogenous OmpR, a transcription factor which activates transcription from the OmpC promoter. Under red light conditions, the activity of the EnvZ histidine kinase domain is inhibited, impeaching transcription from the downstream sequence. </p>
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   <h2 class="title2">Conception</h2>
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<p class="texte">The following construction was designed:<br>
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  <p class="texte"><i>Description des conditions d'expérimentation</i></p>
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   <p class="texteprotocol"><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/notebook/protocols/charac_recomb">In vitro recombinase characterization protocol</a></p>
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   <img src="https://static.igem.org/mediawiki/2013/5/52/Red_sensor80.png" class="imgcontent" />
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In order to mimic the real behavior of the red light sensor system in the <i>E. calculus</i> design, Cph8 gene was placed under the control of the pTet promoter, the general inducer system. The transformed strain was supposed to express the modified RFP while in the dark. In red light conditions, expression was supposedly promoted, except when aTc was added in the media.</p>
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<img style="width:20px"  src="https://static.igem.org/mediawiki/2013/2/23/Top_arrow.png"class="imgcontent2" /><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/results">Back to the top</a></p>
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   <h2 class="title2">Result</h2>
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   <h2 class="faq" title="Click to open the FAQ">   In vivo</h2>
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  <img style="width:340px; float:left;" src="https://static.igem.org/mediawiki/2013/2/20/XOR1_petit.jpg" class="imgcontent" />
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  <p class="texte"><i>Description des conditions d'expérimentation</i></p>
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<p class="texte">We obtain the previous construction to characterize the red light sensor system.<br>
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  <p class="texteprotocol"><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/notebook/protocols/charac_recomb">In vitro recombinase characterization protocol</a></p>
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Used parts are available here:<br>
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- <a href="http://parts.igem.org/Part:BBa_K608002" target="_blank">Strong promoter and strong rbs</a><br>  
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<img style="width:20px"  src="https://static.igem.org/mediawiki/2013/2/23/Top_arrow.png"class="imgcontent2" /><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/results">Back to the top</a></p>
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- <a href="http://parts.igem.org/Part:BBa_I15008" target="_blank">Ho1</a><br>
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- <a href="http://parts.igem.org/Part:BBa_K081017" target="_blank">PcyA</a><br>
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- <a href="http://parts.igem.org/Part:BBa_P0440" target="_blank">TetR</a><br>
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- <a href="http://parts.igem.org/Part:BBa_R0082" target="_blank">pOmpC promoter</a><br>
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- <a href="http://parts.igem.org/Part:BBa_K081014" target="_blank">rfp</a><br>
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- <a href="http://parts.igem.org/Part:BBa_R0040" target="_blank">ptet</a><br>
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- <a href="http://parts.igem.org/Part:BBa_K592018" target="_blank">Cph8</a><br>
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</p>
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  <p class="texteprotocol"><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/notebook/protocols/charac_recomb">In vitro recombinase characterization protocol</a></p>
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<img style="width:20px"  src="https://static.igem.org/mediawiki/2013/2/23/Top_arrow.png"class="imgcontent2" /><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/results">Back to the top</a></p>
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  <h2 class="faq" title="Click to open the FAQ">  In vivo</h2>
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  <img style="width:340px; float:left;" src="https://static.igem.org/mediawiki/2013/2/20/XOR1_petit.jpg" class="imgcontent" />
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  <img style="width:340px; float:right;" src="https://static.igem.org/mediawiki/2013/6/6b/XOR1_bio_80.jpg" class="imgcontent" />
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  <p class="texteprotocol"><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/notebook/protocols/charac_recomb">In vitro recombinase characterization protocol</a></p>
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<img style="width:20px"  src="https://static.igem.org/mediawiki/2013/2/23/Top_arrow.png"class="imgcontent2" /><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/results">Back to the top</a></p>
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<h3 class="title3">Red Light Sensor</h3>
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  <h2 class="faq" title="Click to open the FAQ">  In vitro</h2>
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  <img style="width:340px; float:left;" src="https://static.igem.org/mediawiki/2013/2/20/XOR1_petit.jpg" class="imgcontent" />
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  <p class="texte"><i>Description des conditions d'expérimentation</i></p>
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  <p class="texteprotocol"><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/notebook/protocols/charac_recomb">In vitro recombinase characterization protocol</a></p>
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<img style="width:20px"  src="https://static.igem.org/mediawiki/2013/2/23/Top_arrow.png"class="imgcontent2" /><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/results">Back to the top</a></p>
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  <h2 class="faq" title="Click to open the FAQ">  In vivo</h2>
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  <img style="width:340px; float:left;" src="https://static.igem.org/mediawiki/2013/2/20/XOR1_petit.jpg" class="imgcontent" />
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  <p class="texteprotocol"><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/notebook/protocols/charac_recomb">In vitro recombinase characterization protocol</a></p>
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<img style="width:20px"  src="https://static.igem.org/mediawiki/2013/2/23/Top_arrow.png"class="imgcontent2" /><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/results">Back to the top</a></p>
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  <h2 class="faq" title="Click to open the FAQ">  In vitro</h2>
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  <img style="width:340px; float:left;" src="https://static.igem.org/mediawiki/2013/2/20/XOR1_petit.jpg" class="imgcontent" />
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  <p class="texteprotocol"><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/notebook/protocols/charac_recomb">In vitro recombinase characterization protocol</a></p>
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<img style="width:20px"  src="https://static.igem.org/mediawiki/2013/2/23/Top_arrow.png"class="imgcontent2" /><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/results">Back to the top</a></p>
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  <h2 class="faq" title="Click to open the FAQ">  In vivo</h2>
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  <p class="texte"><i>Description des conditions d'expérimentation</i></p>
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  <p class="texteprotocol"><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/notebook/protocols/charac_recomb">In vitro recombinase characterization protocol</a></p>
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<img style="width:20px"  src="https://static.igem.org/mediawiki/2013/2/23/Top_arrow.png"class="imgcontent2" /><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/results">Back to the top</a></p>
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<h3 class="title3">Pol T7</h3>
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  <h2 class="faq" title="Click to open the FAQ">  In vitro</h2>
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  <p class="texteprotocol"><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/notebook/protocols/charac_recomb">In vitro recombinase characterization protocol</a></p>
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<img style="width:20px"  src="https://static.igem.org/mediawiki/2013/2/23/Top_arrow.png"class="imgcontent2" /><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/results">Back to the top</a></p>
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  <img style="width:340px; float:left;" src="https://static.igem.org/mediawiki/2013/2/20/XOR1_petit.jpg" class="imgcontent" />
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  <p class="texteprotocol"><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/notebook/protocols/charac_recomb">In vitro recombinase characterization protocol</a></p>
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<img style="width:20px"  src="https://static.igem.org/mediawiki/2013/2/23/Top_arrow.png"class="imgcontent2" /><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/results">Back to the top</a></p>
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<h3 class="title3">General inductor</h3>
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  <h2 class="faq" title="Click to open the FAQ">  In vivo</h2>
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<img src="https://static.igem.org/mediawiki/2013/d/d4/General_inducer_notebook_-_680px.png" class="imgcontent" />
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  <p class="texte">The couple TetR-pTet system construction is a first step to characterize the entire system. A constitutive promoter (BBa_J23116) was assemble to tetR and then assemble to pTet-rbs-RFP-term (Bba_I13521). pTet is a regulator constitutively ON that expresses the mRFP1 protein. TetR is an inducer that binds to pTet promoter and thus, represses expression the downstream system. Supply of tetracycline or its analog aTc (anhydrotetracycline) is known to bind to tetR and invert the operation (inhibits expression of mRFP in this case).<br>
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After 18 hours, clones containing this assembly (BBa_J23116-TetR-pTet-RFP) show a leaky basal expression of mRFP. We suppose that the promoter was too weak to express TetR in large quantity. Assembly of a stronger promoter could improve the system and lock the response to an ON/OFF response. <br>
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Besides, an experience was done to analyze the effect of the aTc inducer. Result show a visible induction of the red fluorescent protein expression by addition of aTC (60 ng/mL).</p>
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<img src="https://static.igem.org/mediawiki/2013/e/e1/Induct_gen_pfaible.png" class="imgcontent" />
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<img style="width:20px"  src="https://static.igem.org/mediawiki/2013/2/23/Top_arrow.png"class="imgcontent2" /><a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/results">Back to the top</a></p>
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  <h2 class="title2">Discussion</h2>
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<p class="texte">We succeeded in cloning the <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1132017" target="_blank">construction</a> in pSB1C3, but did not characterize it due to lack of time. The final biobrick must be used in an <i>E. coli</i> deficient in EnvZ. The delta EnvZ strain was obtained from the Paris Bettencourt team and has been already described in many other iGEM projects.
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Latest revision as of 01:05, 5 October 2013

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Results - Red Light Sensor Characterization

Objective

Characterize the red light sensor system using Cph8 with its promoter pOmpc. Reminder: in darkness, the EnvZ histidine kinase of Cph8 phosphorylates endogenous OmpR, a transcription factor which activates transcription from the OmpC promoter. Under red light conditions, the activity of the EnvZ histidine kinase domain is inhibited, impeaching transcription from the downstream sequence.

Conception

The following construction was designed:

In order to mimic the real behavior of the red light sensor system in the E. calculus design, Cph8 gene was placed under the control of the pTet promoter, the general inducer system. The transformed strain was supposed to express the modified RFP while in the dark. In red light conditions, expression was supposedly promoted, except when aTc was added in the media.

Result

We obtain the previous construction to characterize the red light sensor system.

Used parts are available here:
- Strong promoter and strong rbs
- Ho1
- PcyA
- TetR
- pOmpC promoter
- rfp
- ptet
- Cph8

Discussion

We succeeded in cloning the construction in pSB1C3, but did not characterize it due to lack of time. The final biobrick must be used in an E. coli deficient in EnvZ. The delta EnvZ strain was obtained from the Paris Bettencourt team and has been already described in many other iGEM projects.

Retrieved from "http://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/results/red_sensor"