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Team:INSA Toulouse/contenu/lab practice/results/red sensor
From 2013.igem.org
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<h2 class="title2">Objective</h2> | <h2 class="title2">Objective</h2> | ||
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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> | 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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<h2 class="title2">Conception</h2><br> | <h2 class="title2">Conception</h2><br> | ||
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The following construction was designed:<br> | The following construction was designed:<br> | ||
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<img src="https://static.igem.org/mediawiki/2013/5/52/Red_sensor80.png" class="imgcontent" /> | <img src="https://static.igem.org/mediawiki/2013/5/52/Red_sensor80.png" class="imgcontent" /> | ||
<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> | <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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| - | + | <h2 class="title2">Result</h2> | |
| - | <h2 class="title2">Result</h2 | + | |
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We obtain the previous construction to characterize the red light sensor system.<br> | We obtain the previous construction to characterize the red light sensor system.<br> | ||
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- <a href="http://parts.igem.org/Part:BBa_K592018">Cph8</a><br> | - <a href="http://parts.igem.org/Part:BBa_K592018">Cph8</a><br> | ||
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| + | <h2 class="title2">Discussion</h2> | ||
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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. | |
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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> | <br> | ||
<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> | <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> | ||
Revision as of 19:36, 3 October 2013
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
