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

From 2013.igem.org

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   <h1 class="title1">Results - Red Light Sensor Characterization</h1>
    
    
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   <h2 class="title2">Objective</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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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. <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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  <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">AND1</h3>
 
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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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   <h2 class="title2">Conception</h2><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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  <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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The following construction was designed:<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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<h3 class="title3">Red Light Sensor</h3>
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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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<p class="texte"> Cph8 gene was under the control of the pTet promoter, the general inducer system to mimic the real behavior of the red light sensor system in the E. calculus design (https://2013.igem.org/Team:INSA_Toulouse). 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.<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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  <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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  <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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   <h2 class="title2">Result</h2><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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  <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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We obtain the previous construction to characterize the red light sensor system.<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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  <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">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">Ho1</a><br>
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- <a href="http://parts.igem.org/Part:BBa_K081017">PcyA</a><br>
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- <a href="http://parts.igem.org/Part:BBa_P0440">TetR</a><br>
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- <a href="http://parts.igem.org/Part:BBa_R0082">pOmpC promoter</a><br>
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- <a href="http://parts.igem.org/Part:BBa_K081014">rfp</a><br>
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- <a href="http://parts.igem.org/Part:BBa_R0040">ptet</a><br>
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- <a href="http://parts.igem.org/Part:BBa_K592018">Cph8</a><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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<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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  <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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  <h2 class="title2">Discussion</h2><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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We succeeded in cloning the <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1132017">construction</a>, but did not characterize it due to lack of time. The final biobrick must be used in an E. coli deficient in wild-type EnvZ. The delta EnvZ strain was obtained from the Paris Bettencourt team and has been already described by many other iGEM projects. <br>
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<img src="https://static.igem.org/mediawiki/2013/e/e1/Induct_gen_pfaible.png" class="imgcontent" />
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<a href="http://www.nature.com/nature/journal/v438/n7067/abs/nature04405.html">http://www.nature.com/nature/journal/v438/n7067/abs/nature04405.html</a><br>
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Revision as of 19:35, 3 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:

Cph8 gene was under the control of the pTet promoter, the general inducer system to mimic the real behavior of the red light sensor system in the E. calculus design (https://2013.igem.org/Team:INSA_Toulouse). 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, but did not characterize it due to lack of time. The final biobrick must be used in an E. coli deficient in wild-type EnvZ. The delta EnvZ strain was obtained from the Paris Bettencourt team and has been already described by many other iGEM projects.

http://www.nature.com/nature/journal/v438/n7067/abs/nature04405.html

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