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Team:INSA Toulouse/contenu/project/biological construction/input
From 2013.igem.org
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| - | <h1 class="title1">Biological | + | <h1 class="title1">Biological Modules: Input</h1> |
| - | <h2 class="title2"> | + | <h2 class="title2"></h2> |
| - | <p class="texte">For the input, | + | <p class="texte">For the input, we needed a signal that could easily represents "ON" and "OFF" states. Light came as a natural solution because it is easily switchable to "ON" and "OFF" states and color can be varied to represent several inputs (A and B). |
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| + | <br> <br> In the final <i>E. calculus</i> design, each light sensor will promote the transcription of a specific recombinase (TP901 for the red sensor and Bxb1 for the blue sensor). </p> | ||
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| - | <p class=" | + | <p class="texte"><span class="spantitle">Blue light system </span></br> |
| - | YF1 is the fusion of LOV protein | + | <p class="texteleft"> <i> General principle</i> |
| - | <img src="https://static.igem.org/mediawiki/2013/ | + | <br> The blue light sensor uses the couple YF1-FixJ/FixK2 promoter. This setup was already used by the iGEM11_Uppsala-Sweden team and also described in the litterature (<a href="http://www.ncbi.nlm.nih.gov/pubmed/19109976" target="_blank"> Andreas Möglich et al. 2009. Design and Signaling Mechanism of Light-Regulated Histidine Kinases.</a>). YF1 is the fusion of the LOV protein with an histidine kinase. In the absence of light, YF1 can activate FixJ (phosphorylaion) which in return activates the PfixK2 promoter. In the presence of a 480 nm wavelength light, YF1 can no longer phosphorylate FixJ leading to extinction of the PfixK2 promoter. |
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| + | <p class="texteright"> <i> Blue light module characterization </i> | ||
| + | <br>To characterize the blue light sensor, a modified RFP was used as the output instead of the recombinase Bxb1. In darkness, RFP is supposed to be expressed whereas in the presence of blue light, no RFP should be produced. Results are described on <a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/results/blue_sensor">this page</a>. | ||
| + | </p> | ||
| + | <img style="width:700px;" src="https://static.igem.org/mediawiki/2013/thumb/3/35/Scheme_blue_sensor.jpg/800px-Scheme_blue_sensor.jpg" class="imgcontentright" /> | ||
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| - | <p class=" | + | <p class="texte"><span class="spantitle">Red light system</span></br> |
| - | + | <p class="texteleft"><i>General principle</i> | |
| - | + | <br> The red light sensor uses CpH8, a chimeric receptor interacting with the promoter OmpC. CpH8 is the fusion of the PCB photoreceptor with the EnvZ histidine kinase. It is therefore essential to use a wild-type EnvZ deficient <i>E. coli</i>. The biosynthesis of PCB requires also two other genes : ho1 and PcyA. In the absence of light, Cph8 is phosphylated and can activate the OmpC promoter. In the presence of a 660 nm wavelength light, CpH8 is no longer phosphorylated and can not activate the OmpC promoter. (<a href="http://www.ncbi.nlm.nih.gov/pubmed/16306980" target="_blank">Levskaya A. and al. 2005. Synthetic biology: engineering Escherichia coli to see light.</a>) </p> | |
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| + | <p class="texteright"><i>Red light system characterization</i> | ||
| + | <br> <br> The system is similar to the blue light sensor and a modified RFP was used as the output instead of the recombinase TP901. In darkness, RFP is supposed to be expressed whereas in presence of red light, no RFP is produced. We obtained the final construction but had not enough time to characterize it. More details here: <a href="https://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/results/red_sensor"> Results</a></p> | ||
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| + | <img style="width:700px;" src="https://static.igem.org/mediawiki/2013/5/5a/Scheme_red_sensor.jpg" class="imgcontentleft" /> | ||
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Latest revision as of 18:01, 4 October 2013
Biological Modules: Input
For the input, we needed a signal that could easily represents "ON" and "OFF" states. Light came as a natural solution because it is easily switchable to "ON" and "OFF" states and color can be varied to represent several inputs (A and B).
In the final E. calculus design, each light sensor will promote the transcription of a specific recombinase (TP901 for the red sensor and Bxb1 for the blue sensor).
Blue light system
General principle
The blue light sensor uses the couple YF1-FixJ/FixK2 promoter. This setup was already used by the iGEM11_Uppsala-Sweden team and also described in the litterature ( Andreas Möglich et al. 2009. Design and Signaling Mechanism of Light-Regulated Histidine Kinases.). YF1 is the fusion of the LOV protein with an histidine kinase. In the absence of light, YF1 can activate FixJ (phosphorylaion) which in return activates the PfixK2 promoter. In the presence of a 480 nm wavelength light, YF1 can no longer phosphorylate FixJ leading to extinction of the PfixK2 promoter.
Blue light module characterization
To characterize the blue light sensor, a modified RFP was used as the output instead of the recombinase Bxb1. In darkness, RFP is supposed to be expressed whereas in the presence of blue light, no RFP should be produced. Results are described on this page.
Red light system
General principle
The red light sensor uses CpH8, a chimeric receptor interacting with the promoter OmpC. CpH8 is the fusion of the PCB photoreceptor with the EnvZ histidine kinase. It is therefore essential to use a wild-type EnvZ deficient E. coli. The biosynthesis of PCB requires also two other genes : ho1 and PcyA. In the absence of light, Cph8 is phosphylated and can activate the OmpC promoter. In the presence of a 660 nm wavelength light, CpH8 is no longer phosphorylated and can not activate the OmpC promoter. (Levskaya A. and al. 2005. Synthetic biology: engineering Escherichia coli to see light.)
Red light system characterization
The system is similar to the blue light sensor and a modified RFP was used as the output instead of the recombinase TP901. In darkness, RFP is supposed to be expressed whereas in presence of red light, no RFP is produced. We obtained the final construction but had not enough time to characterize it. More details here: Results
