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

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                                     <ul>
 
                                     <ul>
 
                                         <li>Three light inducible split proteases were designed and shown to rapidly
 
                                         <li>Three light inducible split proteases were designed and shown to rapidly
−
                                             respond to regulation by blue light stimulation.
+
                                             respond to stimulation with blue light.
 
                                     </ul>
 
                                     </ul>
 
                                 </b></p>
 
                                 </b></p>
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                                 activation of different biological processes. Small molecules and other chemical
 
                                 activation of different biological processes. Small molecules and other chemical
 
                                 signals lack spatial resolution and their temporal resolution is limited by the time
 
                                 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
+
                                 required for cell permeation. In comparison, induction by light as developed by
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                                 the optogenetics offers many advantages. It is fast as well as inexpensive and allows
+
                                 optogenetics offers many advantages. It is fast as well as inexpensive and allows
 
                                 for excellent spatial, temporal and dose-dependent control.
 
                                 for excellent spatial, temporal and dose-dependent control.
 
                             </p>
 
                             </p>
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                                     <p>
 
                                     <p>
 
                                         There is a plethora of various light inducible systems available; however, not
 
                                         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
+
                                         many are applicable to our purpose. Red light induced systems like the PhyB/PIF6 system
 
                                         require an
 
                                         require an
 
                                         additional phytochrome
 
                                         additional phytochrome
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                                         the system can only be induced once
 
                                         the system can only be induced once
 
                                         <x-ref>Niopek2014</x-ref>
 
                                         <x-ref>Niopek2014</x-ref>
−
                                         . As a consequence of this we selected for the purpose of our iGEM project
+
                                         . Therefore, we selected systems responsive to blue light for the purpose of our iGEM project.
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                                        systems responsive to blue light.
+
                                         Of these, the FKF1/GIGANTEA system displays slow association and dissociation rates
−
                                         Of these, FKF1/GIGANTEA displays slow association and dissociation rates
+
 
                                         <x-ref>Guntas2015</x-ref>
 
                                         <x-ref>Guntas2015</x-ref>
 
                                         , making it impractical for fast response purposes, while VVD displays
 
                                         , making it impractical for fast response purposes, while VVD displays
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                                 and in mammalian cells by <a
 
                                 and in mammalian cells by <a
 
                                     href="https://2014.igem.org/Team:Freiburg/Project/The_light_system">Freiburg_2014</a>.
 
                                     href="https://2014.igem.org/Team:Freiburg/Project/The_light_system">Freiburg_2014</a>.
−
                                 As LOV2 is a small photosensory domain from Avena sativa
+
                                 AsLOV2 is a small photosensory domain from <i>Avena sativa</i>
 
                                 phototropin 1 with a C-terminal Jα helix. The Jα helix is caged in darkness but unfolds
 
                                 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
 
                                 upon blue light (< 500 nm) photoexcitation, which is crucial for phototropin
−
                                 signaling.
+
                                 signalling.
 
                             </p>
 
                             </p>
  
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                                 <h3 style="clear:both;"><span id="lov" class="section colorize">&nbsp;</span>LOVpep and ePDZb
 
                                 <h3 style="clear:both;"><span id="lov" class="section colorize">&nbsp;</span>LOVpep and ePDZb
 
                                 </h3>
 
                                 </h3>
−
                                 <p>For initial testing and characterization of the system, we fused the LOVpep and ePDZb
+
                                 <p>For initial testing and characterization of the system, we fused LOVpep and ePDZb
 
                                     <x-ref>Muller2014</x-ref>
 
                                     <x-ref>Muller2014</x-ref>
 
                                     to corresponding segments of the
 
                                     to corresponding segments of the
 
                                     <a href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Reporters#cle">split
 
                                     <a href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Reporters#cle">split
−
                                         firefly luciferase</a>. We tested different positions of the split protein on
+
                                         firefly luciferase</a> (<ref>1</ref>). We tested different positions of the split protein on
 
                                     the PDZ domain,
 
                                     the PDZ domain,
 
                                     while the split protein was kept at the N-terminus of the LOVpep domain due to the
 
                                     while the split protein was kept at the N-terminus of the LOVpep domain due to the
−
                                     importance of the C-terminal peptide epitope (
+
                                     importance of the C-terminal peptide epitope. We also tested different ratios of both parts of the dimerizing system to determine the optimal conditions for further experiments (<ref>2</ref>B).
−
                                    <ref>1</ref>
+
−
                                    ).
+
 
                                 </p>
 
                                 </p>
 
                                 <div style="margin-left:auto; margin-right:auto; width:70%">
 
                                 <div style="margin-left:auto; margin-right:auto; width:70%">
 
                                     <figure data-ref="1">
 
                                     <figure data-ref="1">
 
                                         <img src="https://static.igem.org/mediawiki/2016/3/39/T--Slovenia--S.4.8.1.png">
 
                                         <img src="https://static.igem.org/mediawiki/2016/3/39/T--Slovenia--S.4.8.1.png">
−
                                         <figcaption><b>LOVpep/ePDZ light inducible system fused to the split firefly
+
                                         <figcaption><b>The LOVpep/ePDZ light inducible system fused to the split firefly
 
                                             luciferase.</b><br/></figcaption>
 
                                             luciferase.</b><br/></figcaption>
 
                                     </figure>
 
                                     </figure>
 
                                 </div>
 
                                 </div>
−
                                 <p style="clear:both">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>B).
+
                                 <p>As luciferase activity was highest with split luciferase on the N-terminus of the
−
                                </p>
+
                                     ePDZ domain and a 1:3 ratio of the LOVpep:ePDZ constructs, all subsequent experiments were
−
                                 <p>As the measured signal was the highest with split luciferase on the N-terminus of the
+
                                     performed with this conditions. An important feature for real life applications is 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
 
                                     ability of the system to be stimulated multiple times. Therefore, repeated
 
                                     association
 
                                     association
−
                                     and dissociation was tested in the real time, by adding luciferin to the medium and
+
                                     and dissociation was tested in real time, by adding luciferin to the medium and
−
                                     measuring bioluminescence upon induction by light (
+
                                     measuring luciferase activity upon induction by light (
 
                                     <ref>2</ref>
 
                                     <ref>2</ref>
−
                                     C). The system showed
+
                                     C). The system exhibited
 
                                     a delayed, but successful induction the first time, but the second induction was
 
                                     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
+
                                     much weaker. These results indicate that the LOVpep/ePDZ system in this setup could
 
                                     not be
 
                                     not be
−
                                     induced more than once, so we decided to test another system.
+
                                     induced more than once, so we decided to test an additional system.
 
                                 </p>
 
                                 </p>
 
                                 <div style="float:left; width:100%">
 
                                 <div style="float:left; width:100%">
 
                                     <figure data-ref="2">
 
                                     <figure data-ref="2">
 
                                         <img src="https://static.igem.org/mediawiki/2016/5/59/T--Slovenia--4.9.1.png">
 
                                         <img src="https://static.igem.org/mediawiki/2016/5/59/T--Slovenia--4.9.1.png">
−
                                         <figcaption><b>LOVpep/ePDZ photoreceptors linked to split luciferase
+
                                         <figcaption><b>LOVpep/ePDZ photoreceptors linked to the split luciferase
 
                                             reporter.</b><br/>
 
                                             reporter.</b><br/>
 
                                             <p style="text-align:justify">(A) Schematic representation of the
 
                                             <p style="text-align:justify">(A) Schematic representation of the
−
                                                 light-inducible interaction between proteins containing ePDZ and LOVpep
+
                                                 light-inducible interaction between proteins containing the ePDZ and LOVpep
 
                                                 domains. (B) Light inducible reporter with split
 
                                                 domains. (B) Light inducible reporter with split
−
                                                 luciferase at the N-terminus of ePDZ domain (nLuc:ePDZb) responded to
+
                                                 luciferase at the N-terminus of the ePDZ domain (nLuc:ePDZb) responded to
−
                                                 light more efficiently than ePDZ:nLuc. Schematic representation of
+
                                                 light more efficiently than ePDZ:nLuc. Corresponding schematic representations of
 
                                                 different arrangements
 
                                                 different arrangements
−
                                                 of ePDZ to split firefly luciferase (nLuc). After induction the cells
+
                                                 of ePDZ fused to the N-terminal segment of split firefly luciferase (nLuc) are shown above the graph. After induction with blue light at 460nm the cells
 
                                                 transfected with LOVpep/ePDZ reporter system were lysed and
 
                                                 transfected with LOVpep/ePDZ reporter system were lysed and
−
                                                 bioluminescence was measured
+
                                                 luciferase activity was determined
−
                                                 with dual luciferase assay. (D) LOVpep/ePDZ reporter reacted to light
+
                                                 with the dual luciferase assay. (C) The LOVpep/ePDZ system responded to light stimulation
 
                                                 only once. Following the addition of luciferin to the medium the cells
 
                                                 only once. Following the addition of luciferin to the medium the cells
−
                                                 were induced and
+
                                                 were induced or
 
                                                 left in the dark for indicated periods.
 
                                                 left in the dark for indicated periods.
 
                                             </p>
 
                                             </p>
Line 248: Line 243:
 
                                 <p>As it has previously been shown on the example of split Cre recombinase
 
                                 <p>As it has previously been shown on the example of split Cre recombinase
 
                                     <x-ref>Kennedy2010</x-ref>
 
                                     <x-ref>Kennedy2010</x-ref>
−
                                     the CRY2-CIB1 interaction seems to be a suitable tool for the
+
                                     the CRY2/CIB1 interaction upon excitation with blue light seems to be a suitable tool for the
 
                                     reconstitution of split proteins, allowing for spatial, temporal and dose-dependent
 
                                     reconstitution of split proteins, allowing for spatial, temporal and dose-dependent
−
                                     optical control of protein dimerization. CRY2 system has also been sued before at
+
                                     optical control of protein dimerization. The CRY2/CIB1 system has also been used before at
 
                                     iGEM:
 
                                     iGEM:
 
                                     <a href="https://2012.igem.org/Team:Duke/Project">Duke 2012</a>, <a
 
                                     <a href="https://2012.igem.org/Team:Duke/Project">Duke 2012</a>, <a
Line 264: Line 259:
 
                                     <div class="content">
 
                                     <div class="content">
 
                                         <p>
 
                                         <p>
−
                                             CRY2 is a cryptochrome, originating from Arabidopsis thaliana and is a blue
+
                                             CRY2 is a cryptochrome, originating from <i>Arabidopsis thaliana</i> and is a blue
 
                                             light–absorbing photosensor that binds a helix-loop-helix DNA-binding
 
                                             light–absorbing photosensor that binds a helix-loop-helix DNA-binding
 
                                             protein CIB1 in
 
                                             protein CIB1 in
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                                     <figure data-ref="4">
 
                                     <figure data-ref="4">
 
                                         <img src="https://static.igem.org/mediawiki/2016/7/76/T--Slovenia--4.9.2.png">
 
                                         <img src="https://static.igem.org/mediawiki/2016/7/76/T--Slovenia--4.9.2.png">
−
                                         <figcaption><b>CRY2PHR/CIBN light inducible receptor linked with split
+
                                         <figcaption><b>CRY2PHR/CIBN light inducible receptor fused to split
−
                                             luciferase repeatedly responded to light.</b><br/>
+
                                             luciferase responded to light efficiently and repeatedly.</b><br/>
−
                                             <p style="text-align:justify">(A) Response to light depended on
+
                                             <p style="text-align:justify">(A) Response to light depended on the
−
                                                 concentration of CIBN:cLuc. After induction the cells transfected with
+
                                                 amount of the CIBN:cLuc plasmid. After induction the cells transfected with
−
                                                 CIBN:cLuc and CRY2PHR:nLuc were lysed and bioluminescence was measured
+
                                                 the CIBN:cLuc and CRY2PHR:nLuc encoding plasmids were lysed and luciferase activity was determined
−
                                                 with dual luciferase assay.
+
                                                 with the dual luciferase assay.
 
                                                 (B) CRY2PHR light reporter was induced repeatedly. Following the
 
                                                 (B) CRY2PHR light reporter was induced repeatedly. Following the
−
                                                 addition of luciferin to the medium of the cells transfected with
+
                                                 addition of luciferin to the medium, cells transfected with
−
                                                CIBN:cLuc and CRY2PHR:nLuc
+
                                              the CIBN:cLuc and CRY2PHR:nLuc encoding plasmids
−
                                                 (ratio 1:3) were induced and left in the dark for indicated periods.
+
                                                 (ratio 1:3) were induced with blue light at 460nm and left in the dark for indicated periods.
 
                                             </p>
 
                                             </p>
 
                                         </figcaption>
 
                                         </figcaption>
 
                                     </figure>
 
                                     </figure>
 
                                 </div>
 
                                 </div>
−
                                 <p>As the measured signal was the highest with a 1:3 ratio of CRY2PHR/CIBN (
+
                                 <p>As luciferase activity was highest with a 1:3 ratio of CRY2PHR:CIBN constructs (
 
                                     <ref>4</ref>
 
                                     <ref>4</ref>
 
                                     A), all subsequent experiments were performed with this ratio. Next we tested if
 
                                     A), all subsequent experiments were performed with this ratio. Next we tested if
−
                                     this system could be induced repeatedly in real time. The CRY2PHR/CIBN system shows
+
                                     this system could be induced repeatedly in real time. The CRY2PHR/CIBN system showed a
−
                                     maximum activity after 2 minutes of induction, returns to background in 10 minutes
+
                                     maximum activity after 2 minutes of induction and dropped to background 10 minutes
 
                                     after
 
                                     after
−
                                     the stimulus is removed, and can be induced repeatedly (
+
                                     the stimulus was removed. The system could be induced repeatedly and reach high levels of activation at each stimulation (
 
                                     <ref>4</ref>
 
                                     <ref>4</ref>
 
                                     B).
 
                                     B).
 
                                 </p>
 
                                 </p>
−
 
+
<figure data-ref="5">
−
                                <div style="float:left; width:100%">
+
−
                                    <figure data-ref="5">
+
 
                                         <img src="https://static.igem.org/mediawiki/2016/9/92/T--Slovenia--4.9.3.png">
 
                                         <img src="https://static.igem.org/mediawiki/2016/9/92/T--Slovenia--4.9.3.png">
−
                                         <figcaption><b>Testing CRY2PHR/CIBN light-inducible system with split
+
                                         <figcaption><b>The CRY2PHR/CIBN mediates reconstitution of orthogonal split TEV protease upon illumination with blue light.</b><br/>
−
                                            protease.</b><br/>
+
                                             <p style="text-align:justify">(A) Schematic representation of the CRY2PHR/CIBN light-inducible system with split protease. In the dark, the CRY2PHR and CIBN proteins do not interact (left), while illumination with blue light results in heterodimerization and reconstitution of split protease (right), which in turn cleaves the cyclic luciferase reporter, resulting in increased luciferase activity. HEK293T cells were transfected with 1:3
−
                                             <p style="text-align:justify">(A) Schematic representation of CRY2PHR/CINB
+
                                                 ratio of plasmids coding for CRY2PHR:CIBN with split TEV protease (B) or split
−
                                                light-inducible system with split protease linked to cyclic luciferase
+
                                                 PPV protease (C). 24 hours after transfection, cells were illuminated with blue light at 460nm for indicated periods of time. The cells were lysed and luciferase activity was determined with the dual luciferase assay. Upon illumination with blue light, the protease cleaves only the cyclic reporter with the correct cleavage site (red bars), while the cyclic reporter with the mismatched cleavage site remains uncleaved (white bars).  
−
                                                reporter. Cells were transfected with 1:3
+
                               
−
                                                 ratio of plasmids coding for CRY2PHR:CIBN with split TEVp (B) or split
+
−
                                                 PPVp protease (C). The system was tested with the corresponding cyclic
+
−
                                                reporter
+
−
                                                (red bars), while orthogonality was tested with mismatched cyclic
+
−
                                                reporter (white bars). After indicated periods of induction the cells
+
−
                                                were lysed and
+
−
                                                bioluminescence was measured with dual luciferase assay.
+
 
                                             </p>
 
                                             </p>
 
                                         </figcaption>
 
                                         </figcaption>
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                                             src="https://static.igem.org/mediawiki/2016/4/44/T--Slovenia--4.9.4.png">
 
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Revision as of 13:30, 19 October 2016

Light dependent mediator

Split proteases Light dependent
mediator Achievements Introduction LOVpep & ePDZb CRY2-CIB1 Light inducible
protease Logic

nbsp;Light-dependent input signal and proteases - in progress TL

  • Three light inducible split proteases were designed and shown to rapidly respond to stimulation with blue light.

 

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 cell permeation. In comparison, induction by light as developed by optogenetics offers many advantages. It is fast as well as inexpensive and allows for excellent spatial, temporal and dose-dependent control.

Further explanation ...

There is a plethora of various light inducible systems available; however, not many are applicable to our purpose. Red light induced systems like the PhyB/PIF6 system require an additional phytochrome Zimmerman2016 , while the UV light used to induce some systems like UVR Schmidt2015 might be toxic to the cells and the system can only be induced once Niopek2014 . Therefore, we selected systems responsive to blue light for the purpose of our iGEM project. Of these, the FKF1/GIGANTEA system displays slow association and dissociation rates Guntas2015 , making it impractical for fast response purposes, while VVD displays fast response, but is a homodimer Zhang2015 , making it unsuitable for successful heterodimerization of split enzymes.


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 signalling.

Further explanation ...

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 Stricklandetal.2012 . This system has already been used for iGEM projects ( Freiburg_2014).

nbsp;Results

 LOVpep and ePDZb

For initial testing and characterization of the system, we fused LOVpep and ePDZb Muller2014 to corresponding segments of the split firefly luciferase (1). 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. We also tested different ratios of both parts of the dimerizing system to determine the optimal conditions for further experiments (2B).

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

As luciferase activity was highest with split luciferase on the N-terminus of the ePDZ domain and a 1:3 ratio of the LOVpep:ePDZ constructs, all subsequent experiments were performed with this conditions. 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 real time, by adding luciferin to the medium and measuring luciferase activity upon induction by light ( 2 C). The system exhibited a delayed, but successful induction the first time, but the second induction was much weaker. These results indicate that the LOVpep/ePDZ system in this setup could not be induced more than once, so we decided to test an additional system.

LOVpep/ePDZ photoreceptors linked to the split luciferase reporter.

(A) Schematic representation of the light-inducible interaction between proteins containing the ePDZ and LOVpep domains. (B) Light inducible reporter with split luciferase at the N-terminus of the ePDZ domain (nLuc:ePDZb) responded to light more efficiently than ePDZ:nLuc. Corresponding schematic representations of different arrangements of ePDZ fused to the N-terminal segment of split firefly luciferase (nLuc) are shown above the graph. After induction with blue light at 460nm the cells transfected with LOVpep/ePDZ reporter system were lysed and luciferase activity was determined with the dual luciferase assay. (C) The LOVpep/ePDZ system responded to light stimulation only once. Following the addition of luciferin to the medium the cells were induced or left in the dark for indicated periods.

 CRY2-CIB1

As it has previously been shown on the example of split Cre recombinase Kennedy2010 the CRY2/CIB1 interaction upon excitation with blue light seems to be a suitable tool for the reconstitution of split proteins, allowing for spatial, temporal and dose-dependent optical control of protein dimerization. The CRY2/CIB1 system has also been used before at iGEM: Duke 2012, Marburg 2013, HNU_China 2014 and ANU_Canberra 2015

Further explanation ...

CRY2 is a cryptochrome, originating from Arabidopsis thaliana and is a blue light–absorbing photosensor that binds a helix-loop-helix DNA-binding protein CIB1 in its photoexcited state. In our system, we used the conserved N-terminal photolyase homology region of CRY2 (CRY2PHR; aa 1-498) that mediates light-responsiveness and the truncated version of the CIB1 protein (CIBN; aa 1-170) without the helix-loop-helix region, which mediates DNA binding Kennedy2010 . Dimerization of CRY2PHR and CIBN can be induced by blue light illumination (460 nm), after which the interaction between CRY2 and CIB1 occurs on a millisecond timescale and can be reversed within minutes by removing the stimulus. This light inducible system has already been implemented for light-dependent transcriptional activation with the Gal4 transcription factor Kennedy2010 , TALEs Konermann2013 and the CRISPR/Cas9 system He2015 . Another application of the system is induction of whole-protein dimerization Wend2014 or split protein dimerization Lin2012 .


We adapted this system for the reconstitution of split luciferase to create a blue-light sensor, which enables easy characterization for further experiments. The N- and C-terminal split fragments of the firefly luciferase were fused to the C-terminus of the CRY2PHR and the CIBN proteins, since this topology has previously been shown to work with the Cre recombinase.

CRY2PHR/CIBN light inducible receptor fused to split luciferase responded to light efficiently and repeatedly.

(A) Response to light depended on the amount of the CIBN:cLuc plasmid. After induction the cells transfected with the CIBN:cLuc and CRY2PHR:nLuc encoding plasmids were lysed and luciferase activity was determined with the dual luciferase assay. (B) CRY2PHR light reporter was induced repeatedly. Following the addition of luciferin to the medium, cells transfected with the CIBN:cLuc and CRY2PHR:nLuc encoding plasmids (ratio 1:3) were induced with blue light at 460nm and left in the dark for indicated periods.

As luciferase activity was highest with a 1:3 ratio of CRY2PHR:CIBN constructs ( 4 A), all subsequent experiments were performed with this ratio. Next we tested if this system could be induced repeatedly in real time. The CRY2PHR/CIBN system showed a maximum activity after 2 minutes of induction and dropped to background 10 minutes after the stimulus was removed. The system could be induced repeatedly and reach high levels of activation at each stimulation ( 4 B).

The CRY2PHR/CIBN mediates reconstitution of orthogonal split TEV protease upon illumination with blue light.

(A) Schematic representation of the CRY2PHR/CIBN light-inducible system with split protease. In the dark, the CRY2PHR and CIBN proteins do not interact (left), while illumination with blue light results in heterodimerization and reconstitution of split protease (right), which in turn cleaves the cyclic luciferase reporter, resulting in increased luciferase activity. HEK293T cells were transfected with 1:3 ratio of plasmids coding for CRY2PHR:CIBN with split TEV protease (B) or split PPV protease (C). 24 hours after transfection, cells were illuminated with blue light at 460nm for indicated periods of time. The cells were lysed and luciferase activity was determined with the dual luciferase assay. Upon illumination with blue light, the protease cleaves only the cyclic reporter with the correct cleavage site (red bars), while the cyclic reporter with the mismatched cleavage site remains uncleaved (white bars).

 Light inducible protease

To implement light as one of the input signals for protease-based signaling pathway or logic functions, we fused the CRY2PHR and the CIBN domains to the N- and C-terminal split domains of 3 different proteases (TEVp, TEVpE and PPVp) ( 5 A) and tested their activity and orthogonality with bioluminescence assays ( 5 B) and western blotting ( 5 C). The bioluminescence assay was based on the split firefly luciferase with cleavage site for either TEVp, TEVpE or PPVp. Cleavage of this reporter results in the luciferase activity reconstitution and thus an increase in the luminescence.

Detection of a substrate cleavage by CRY2PHR/CIBN split protease induced by light.

(A) Schematic representation of CRY2PHR/CIBN light-inducible system with split protease and substrate with protease target sequence. Activity of (B) TEV, (C) PPV and (D) TEV:E proteases were analyzed by Western blot analysis. 24 hours after transfection of cells with plasmids expressing split proteases and protease substrate, the formation of active protease was induced by light. After the indicated time periods cells were immediately lysed and formation of cleaved products were analysed by Western blot using primary antibodies against AU1 tag. Excluding the non-specific upper band, uncleaved samples show only one band, while the cleaved ones show two bands.

The reporter used for the western blot analysis was the luciferase reporter with the appropriate cleavage substrate inserted at a permissible site and an AU1 tag at the N-terminal. Uncleaved luciferase appears as a single band on a western blot, while partial cleavage of luciferase results in two bands (uncleaved at 65 kDa and cleaved at 55 kDa) and complete cleavage results in only the smaller band ( 6 ). We showed that substrates carrying specific target peptide for proteases were cleaved after induction of split protease reporters.

Both methods showed a successful, fast and dose-dependent response. This is the first time split TEV protease has been shown to work as a light inducible system. Also this is the first time TEVpE and PPVp were prepared as split proteins and shown to function in an inducible system.

 References