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</figure> | </figure> | ||
</div> | </div> | ||
− | |||
<p>We tested two orientations of ePDZ (ePDZ fused to split luciferase to N or C terminus) and ratios of the constructs ePDZ vs. cLuc:LOVpep were tested (<ref>2</ref>). | <p>We tested two orientations of ePDZ (ePDZ fused to split luciferase to N or C terminus) and ratios of the constructs ePDZ vs. cLuc:LOVpep were tested (<ref>2</ref>). | ||
</p> | </p> | ||
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</figure> | </figure> | ||
</div> | </div> | ||
+ | <p>As the measured signal was the highest with split luciferase on the N-terminus of the ePDZ domain and a 1:3 ratio of LOVpep:ePDZ, all subsequent experiments were | ||
+ | performed with this ratio. An important feature for real life applications is the ability of the system to be stimulated multiple times. Therefore, repeated association | ||
+ | and dissociation was tested in the real time, by adding luciferin to the medium and measuring bioluminescence upon induction by light (Figure 4.9.1.C.). The system showed | ||
+ | a delayed, but successful induction the first time, but the second induction was much weaker. The results indicated that the LOVpep/ePDZ system in this setup could not be | ||
+ | induced more than once, so we decided to test another system. | ||
+ | </p> | ||
+ | <p>As it has previously been shown on the example of split Cre recombinase <x-ref>Kennedy2010</x-ref> the CRY2-CIB1 interaction seems to be a suitable tool for the | ||
+ | reconstitution of split proteins, allowing for spatial, temporal and dose-dependent optical control of protein dimerization. CRY2 system has also been sued before at iGEM: | ||
+ | <a href="https://2012.igem.org/Team:Duke/Project">Duke 2012</a>, <a href="https://2013.igem.org/Team:Marburg">Marburg 2013</a>, | ||
+ | <a href="https://2014.igem.org/Team:HNU_China/Project"> HNU_China 2014</a> and <a href="https://2015.igem.org/Team:ANU-Canberra">ANU_Canberra 2015</a> | ||
+ | </p> | ||
Revision as of 18:28, 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
We tested two orientations of ePDZ (ePDZ fused to split luciferase to N or C terminus) and ratios of the constructs ePDZ vs. cLuc:LOVpep were tested (2).
As the measured signal was the highest with split luciferase on the N-terminus of the ePDZ domain and a 1:3 ratio of LOVpep:ePDZ, all subsequent experiments were performed with this ratio. An important feature for real life applications is the ability of the system to be stimulated multiple times. Therefore, repeated association and dissociation was tested in the real time, by adding luciferin to the medium and measuring bioluminescence upon induction by light (Figure 4.9.1.C.). The system showed a delayed, but successful induction the first time, but the second induction was much weaker. The results indicated that the LOVpep/ePDZ system in this setup could not be induced more than once, so we decided to test another system.
As it has previously been shown on the example of split Cre recombinase