Difference between revisions of "Team:Slovenia/Protease signaling/Light dependent mediator"

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<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>).
 
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<figure data-ref="2">
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<img onclick="resize(this);" class="ui medium image" src="https://static.igem.org/mediawiki/2016/5/59/T--Slovenia--4.9.1.png" >
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<figcaption><b>LOVpep/ePDZ photoreceptors linked to split luciferase reporter.</b><br/>
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(A) Schematic representation of the light-inducible interaction between proteins containing ePDZ and LOVpep domains. (B) Light inducible reporter with split
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luciferase at the N-terminus of ePDZ domain (nLuc:ePDZb) responded to light more efficiently than ePDZ:nLuc. Schematic representation of different arrangements
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of ePDZ to split firefly luciferase (nLuc). After induction the cells transfected with LOVpep/ePDZ reporter system were lysed and bioluminescence was measured
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with dual luciferase assay. (D) LOVpep/ePDZ reporter reacted to light only once. Following the addition of luciferin to the medium the cells were induced and
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left in the dark for indicated periods.</figcaption>
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Revision as of 18:26, 15 October 2016

Light dependent mediator

Project Achievements Medal requirements Idea

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 Muller2014 to corresponding segments of the split firefly luciferase. 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 (1).

LOVpep/ePDZ light inducible system fused to the split firefly luciferase.

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).

LOVpep/ePDZ photoreceptors linked to split luciferase reporter.
(A) Schematic representation of the light-inducible interaction between proteins containing ePDZ and LOVpep domains. (B) Light inducible reporter with split luciferase at the N-terminus of ePDZ domain (nLuc:ePDZb) responded to light more efficiently than ePDZ:nLuc. Schematic representation of different arrangements of ePDZ to split firefly luciferase (nLuc). After induction the cells transfected with LOVpep/ePDZ reporter system were lysed and bioluminescence was measured with dual luciferase assay. (D) LOVpep/ePDZ reporter reacted to light only once. Following the addition of luciferin to the medium the cells were induced and left in the dark for indicated periods.