Difference between revisions of "Team:Slovenia/Protease signaling/Light dependent mediator"
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<div class = "ui segment"> | <div class = "ui segment"> | ||
<h4>Introduction</h4><br/> | <h4>Introduction</h4><br/> | ||
| − | + | <p>In the recent years, light has been extensively explored as a trigger signal for activation of different biological processes. Small molecules and other chemical | |
| − | + | signals lack spatial resolution and their temporal resolution is limited by the time required for the cell permeation. In comparison, induction by light as developed by | |
| − | + | the optogenetics offers many advantages. It is fast as well as inexpensive and allows for excellent spatial, temporal and dose-dependent control. | |
| − | + | </p> | |
| − | + | <!--tukej manjka extended text | |
| − | + | There is a plethora of various light inducible systems available; however, not many are applicable to our purpose. Red light induced systems like Phy/PIF require an | |
| − | + | additional phytochrome <x-ref>Zimmerman2016</x-ref>, while the UV light used to induce some systems like UVR <x-ref>Schmidt2015</x-ref> might be toxic to the cells and | |
| − | + | the system can only be induced once <x-ref>Niopek2014</x-ref>. As a consequence of this we selected for the purpose of our iGEM project systems responsive to blue light. | |
| − | + | Of these, FKF1/GIGANTEA displays slow association and dissociation rates <x-ref>Guntas2015</x-ref>, making it impractical for fast response purposes, while VVD displays | |
| − | + | fast response, but is a homodimer <x-ref>Zhang2015</x-ref> (Zhang & Cui, 2015), making it unsuitable for successful heterodimerization of split enzymes. | |
| − | + | --> | |
| − | + | <p>Initially we decided to test the LOVpep/ePDZ system. This system has been used previously at iGEM, by <a href="https://2009.igem.org/Team:EPF-Lausanne/LOVTAP"> | |
| − | + | EPF_Lausanne 2009</a>, <a href="https://2011.igem.org/Team:Rutgers/Etch_a_Sketch"> Rutgers 2011</a> and <a href="https://2012.igem.org/Team:Rutgers/BEAS">Rutgers 2012</a> | |
| − | + | and in mammalian cells by <a href="https://2014.igem.org/Team:Freiburg/Project/The_light_system">Freiburg_2014</a>. AsLOV2 is a small photosensory domain from Avena sativa | |
| − | + | phototropin 1 with a C-terminal Jα helix. The Jα helix is caged in darkness but unfolds upon blue light (< 500 nm) photoexcitation, which is crucial for phototropin | |
| − | + | signaling. | |
| − | + | </p> | |
| − | + | <!--tukej manjka extended text | |
| − | + | A photosensor has been prepared by engineering the AsLOV2 domain to contain a peptide epitope SSADTWV on the C-terminus of the Jα helix (LOVpep), binding an engineered | |
| − | + | Erbin PDZ domain (ePDZ) upon blue light stimulation <x-ref> Stricklandetal.2012 </x-ref>. This system has already been used for iGEM projects ( | |
| + | <a href="https://2014.igem.org/Team:Freiburg">Freiburg_2014</a>). | ||
| + | --> | ||
| + | <h4>Results:<h4> | ||
| + | |||
| + | <p>For initial testing and characterization of the system, we fused the LOVpep and ePDZb <x-ref>Muller2014</x-ref> to corresponding segments of the <a href=«https://2016.igem.org/Team:Slovenia/Protease_signaling/Reporters«>split firefly luciferase</a>. We tested different positions of the split protein on the PDZ domain, while the split protein was kept at the N-terminus of the LOVpep domain due to the importance of the C-terminal peptide epitope (<ref>1</ref>).</p> | ||
| + | |||
</div> | </div> | ||
</div> | </div> | ||
Revision as of 18:17, 15 October 2016
nbsp;Light-depended mediator
Achivements
We designed and successfully tested three light inducible split proteases: CIBN/CRY2PHR light inducible split TEV protease, CIBN/CRY2PHR light inducible split PPVp and CIBN/CRY2PHR light inducible split TEVpE.
Introduction
In the recent years, light has been extensively explored as a trigger signal for activation of different biological processes. Small molecules and other chemical signals lack spatial resolution and their temporal resolution is limited by the time required for the cell permeation. In comparison, induction by light as developed by the optogenetics offers many advantages. It is fast as well as inexpensive and allows for excellent spatial, temporal and dose-dependent control.
Initially we decided to test the LOVpep/ePDZ system. This system has been used previously at iGEM, by EPF_Lausanne 2009, Rutgers 2011 and Rutgers 2012 and in mammalian cells by Freiburg_2014. AsLOV2 is a small photosensory domain from Avena sativa phototropin 1 with a C-terminal Jα helix. The Jα helix is caged in darkness but unfolds upon blue light (< 500 nm) photoexcitation, which is crucial for phototropin signaling.
Results:
For initial testing and characterization of the system, we fused the LOVpep and ePDZb
