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

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     <title>Split proteases</title>
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     <title>Light dependent mediator</title>
 
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                 <a href = "//2016.igem.org/Team:Slovenia">
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                     <img class="ui medium sticky image" src="//2016.igem.org/wiki/images/d/d1/T--Slovenia--logo.png">
 
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                     <a class="item" href="//2016.igem.org/Team:Slovenia/Protease_signaling/Orthogonality">
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                     <a class="item" href="//2016.igem.org/Team:Slovenia/Protease_signaling/Split_proteases">
 
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                         <b>Orthogonality</b>
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                         <b>Split proteases</b>
 
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                     </a>
 
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                         <b>Motivation</b>
 
                         <b>Motivation</b>
 
                     </a>
 
                     </a>
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                     <a class="item" href="#res" style="margin-left: 10%">
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                     <a class="item" href="#lov" style="margin-left: 10%">
 
                         <i class="selected radio icon"></i>
 
                         <i class="selected radio icon"></i>
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                         <b>Results</b>
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                         <b>LOVpep & ePDZb</b>
 
                     </a>
 
                     </a>
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                     <a class="item" href="//2016.igem.org/Team:Slovenia/Protease_signaling/Light_dependent_mediator">
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                    <a class="item" href="#cry" style="margin-left: 10%">
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                        <i class="selected radio icon"></i>
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                        <b>CRY2-CIB1</b>
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                    </a>
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                    <a class="item" href="#lig" style="margin-left: 10%">
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                        <i class="selected radio icon"></i>
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                        <b>Light inducible</b>
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                        <br/>
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                        <b style="margin-left: 12%">protease</b>
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                    </a>
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                     <a class="item" href="//2016.igem.org/Team:Slovenia/Protease_signaling/Logic">
 
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                         <i class="chevron circle right icon"></i>
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                         <b>Light-dependent mediator</b>
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                         <b>Logic</b>
 
                     </a>
 
                     </a>
  
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                         <div>
 
                         <div>
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                             <h1 class = "ui left dividing header"><span id = "ach" class="section">&nbsp;</span>Split orthogonal proteases</h1>
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                             <h1 class="ui left dividing header"><span id="ach" class="section">nbsp;</span>Light-dependent
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                             <div class = "ui segment" style = "background-color: #ebc7c7; ">
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                                input signal and proteases</h1>
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                                 <p><b><ul>
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                             <div class="ui segment" style="background-color: #ebc7c7; ">
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                                    <li>Four new active split site-specific proteases were designed in addition to the previously published TEVp.
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                                 <p><b>
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                                    <li>The new split proteases were shown to rapidly respond to regulated by induced chemical dimerization.
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                                    <ul>
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                                </ul></b></p>
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                                        <li>Three light inducible split proteases were designed and shown to rapidly
 +
                                            respond to regulation by blue light stimulation.
 +
                                    </ul>
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                                </b></p>
 
                             </div>
 
                             </div>
 
                         </div>
 
                         </div>
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                        <div class="ui segment">
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                            <h4><span class="section" id="mot">&nbsp;</span></h4>
 +
                            <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>
 +
                            <div class="ui styled fluid accordion">
 +
                                <div class="title">
 +
                                    <i class="dropdown icon"></i>
 +
                                    Further explanation ...
 +
                                </div>
 +
                                <div class="content">
 +
                                    <p>
 +
                                        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>
 +
                                        , making it unsuitable for successful heterodimerization of split enzymes.
 +
                                    </p>
 +
                                </div>
 +
                            </div>
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                            <p>
 +
                                <br/>
 +
                                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>.
 +
                                As LOV2 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>
  
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                        <div class = "ui segment">
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                            <div class="ui styled fluid accordion">
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                            <h4><span id = "mot" class="section">&nbsp;</span></h4>
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                                <div class="title">
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                            <p>The split protein system based on the inducible dimerization is an attractive method to regulate the protease activity. Wehr et al. <x-ref>Wehr2006</x-ref> described a
+
                                    <i class="dropdown icon"></i>
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                                split TEVp expressed as two functionally inactive fragments; the N-terminal (1 – 118 aa) and C-terminal (119 – 242 aa) protease fragments (referred to as cTEVp and nTEVp) (<ref>1</ref>).
+
                                    Further explanation ...
−
                                When the two fragments were coexpressed as fusion constructs with adjacent dimerization partners, the split TEVp was able to reconstitute and regain its catalytic activity,
+
                                </div>
−
                                 demonstrating that the activity of split TEVp could be controlled through the ligand induced protein – protein interactions.</p>
+
                                <div class="content">
 +
                                    <p>
 +
                                        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>).
 +
                                    </p>
 +
                                 </div>
 +
                            </div>
 +
                        </div>
  
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                        <h1><span class="section">nbsp;</span>Results</h1>
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                             <div style="float:right; width:40%">
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                        <div class="ui segment">
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                                <figure data-ref="1">
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                             <div>
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                                    <img src="https://static.igem.org/mediawiki/2016/0/00/T--Slovenia--4.4.5.png">
+
                                <h3 style="clear:both;"><span id="lov" class="section">&nbsp;</span>LOVpep and ePDZb
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                                    <figcaption><b>Reconstituted split-TEVp</b><br/>
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                                </h3>
−
                                         <p style="text-align:justify">Model of nTEVp (residues 1 – 118 in blue) and cTEVp (residues 119 – 242 in orange) reconstituted in the active form,
+
                                <p>For initial testing and characterization of the system, we fused the LOVpep and ePDZb
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                                            (from PDB 1LVB).
+
                                    <x-ref>Muller2014</x-ref>
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                                        </p>
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                                    to corresponding segments of the
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                                    </figcaption>
+
                                    <a href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Reporters">split
−
                                </figure>
+
                                        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 style="margin-left:auto; margin-right:auto; width:70%">
 +
                                    <figure data-ref="1">
 +
                                        <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
 +
                                            luciferase.</b><br/></figcaption>
 +
                                    </figure>
 +
                                </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>
 +
                                <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 (
 +
                                    <ref>2</ref>
 +
                                    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>
 +
                                <div style="float:left; width:100%">
 +
                                    <figure data-ref="2">
 +
                                         <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
 +
                                            reporter.</b><br/>
 +
                                            <p style="text-align:justify">(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.
 +
                                            </p>
 +
                                        </figcaption>
 +
                                    </figure>
 +
                                </div>
 
                             </div>
 
                             </div>
  
−
 
+
                             <div>
−
                             <p>Our team hypothesized that the same inducible dimerization approach could also be used with TEVp homologues. We converted all of the tested orthogonal potyviral proteases
+
                                 <h3 style="clear:both;"><span id="cry" class="section">&nbsp;</span>CRY2-CIB1</h3>
−
                                 to split proteases by splitting them at positions corresponding to the position of the previously described split TEV protease. We selected three different types of
+
                                 <p>As it has previously been shown on the example of split Cre recombinase
−
                                 dimerization domains to induce the activity of the split proteases. The first pair of dimerization domains was the rapamycin responsive FKBP/FRB system <x-ref> Banaszynski
+
                                    <x-ref>Kennedy2010</x-ref>
−
                                </x-ref>, which induces dimerization upon ligand binding. The second pair of dimerization domains was the
+
                                    the CRY2-CIB1 interaction seems to be a suitable tool for the
−
                                <a href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Light_dependent_mediator#cry">CRY2PHR/CIBN system</a>, which induces dimerization upon irradiation with blue light.
+
                                    reconstitution of split proteins, allowing for spatial, temporal and dose-dependent
−
                                Finally, our third system for dimerization was designed to respond to a Ca<sup>2+</sup> influx based on the
+
                                    optical control of protein dimerization. CRY2 system has also been sued before at
−
                                <a href="https://2016.igem.org/Team:Slovenia/Mechanosensing/CaDependent_mediator"> calmodulin-M13 interaction </a>, that we used to detect mechanosensing.
+
                                    iGEM:
−
                            </p>
+
                                    <a href="https://2012.igem.org/Team:Duke/Project">Duke 2012</a>, <a
−
                            <div style="float:left; width:60%">
+
                                            href="https://2013.igem.org/Team:Marburg">Marburg 2013</a>,
−
                                 <div style="clear:both" class="ui styled fluid accordion">
+
                                    <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>
 +
                                 <div class="ui styled fluid accordion" style="clear:both;">
 
                                     <div class="title">
 
                                     <div class="title">
 
                                         <i class="dropdown icon"></i>
 
                                         <i class="dropdown icon"></i>
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                                     </div>
 
                                     </div>
 
                                     <div class="content">
 
                                     <div class="content">
−
 
+
                                         <p>
−
                                         <div style="float:right; width:60%">
+
                                             CRY2 is a cryptochrome, originating from Arabidopsis thaliana and is a blue
−
                                             <figure data-ref="4.10.1">
+
                                            light–absorbing photosensor that binds a helix-loop-helix DNA-binding
−
                                                <img src="https://static.igem.org/mediawiki/2016/d/dd/T--Slovenia--4.10.1.png">
+
                                            protein CIB1 in
−
                                                <figcaption><b>Rapamycin and its function.</b><p style="text-align:justify">(A) Chemical structure of rapamycin. Binding sites for FRB and FKBP are shown. (B) Schematic presentation of FKBP and FRB binding to
+
                                            its photoexcited state. In our system, we used the conserved N-terminal
−
                                                    rapamycin<x-ref>Banaszynski</x-ref>
+
                                            photolyase homology region of CRY2 (CRY2PHR; aa 1-498) that mediates
−
                                                </p>
+
                                            light-responsiveness and
−
                                                </figcaption>
+
                                            the truncated version of the CIB1 protein (CIBN; aa 1-170) without the
−
                                             </figure>
+
                                            helix-loop-helix region, which mediates DNA binding
−
                                        </div>
+
                                            <x-ref> Kennedy2010</x-ref>
−
                                        <p>Interactions among different proteins play a key role among all living organisms. Chemically induced dimerization (CID) is one of such interactions,
+
                                            . Dimerization of
−
                                             which allows two different protein domains to dimerize after the addition of a small molecule. The most widely used CID to date is the FKBP/FRB system
+
                                            CRY2PHR and CIBN can be induced by blue light illumination (460 nm), after
−
                                             which heterodimerizes upon rapamycin addition <x-ref> Inobe2016 </x-ref>.
+
                                            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
 +
                                            <x-ref> Kennedy2010</x-ref>
 +
                                            , TALEs
 +
                                            <x-ref>Konermann2013</x-ref>
 +
                                             and the CRISPR/Cas9 system
 +
                                            <x-ref>He2015</x-ref>
 +
                                            . Another application of the system
 +
                                             is induction of whole-protein dimerization
 +
                                            <x-ref>Wend2014</x-ref>
 +
                                            or split protein dimerization
 +
                                             <x-ref>Lin2012</x-ref>
 +
                                            .
 
                                         </p>
 
                                         </p>
−
                                        <p>Rapamycin is a 31-membered macrolide antifungal antibiotic that was first isolated from the Streptomyces hygroscopicus and binds with high affinity to the
 
−
                                            12-kDa FK506 binding protein (FKBP) as well as to a 100-aminoacid domain (E2015 to Q2114) of the mammalian target of rapamycin (mTOR) protein known as the
 
−
                                            FKBP-rapamycin binding domain (FRB) (<ref>4.10.1</ref>) <x-ref>Banaszynski</x-ref>. Besides FKBP/FRB there are also other CID system where small molecules like
 
−
                                            gibberellin <x-ref>Murase</x-ref> and coumermycin <x-ref>Farrar2000</x-ref> are used for induced dimerization.
 
−
                                        </p>
 
−
 
 
                                     </div>
 
                                     </div>
 
                                 </div>
 
                                 </div>
−
                            </div><p style="clear:both"></p>
+
                                <p><br/></p>
−
                        </div>
+
                                <p style="clear:left">We adapted this system for the reconstitution of split luciferase
−
 
+
                                     to create a blue-light sensor, which enables easy characterization for further
−
 
+
                                    experiments.
−
                        <div>
+
                                    The N- and C-terminal split fragments of the firefly luciferase were fused to the
−
                            <h1><span id = "res" class="section">&nbsp;</span>Results</h1>
+
                                     C-terminus of the CRY2PHR and the CIBN proteins, since this topology has previously
−
                            <div class = "ui segment">
+
                                    been shown to work with the Cre recombinase.
−
                                <p>We tested the full set of four orthogonal proteases with the rapamycin inducible system by measuring their activity with the cycLuc reporter. Increasing luciferase
+
−
                                     activity was detected correlating with the amount of the transfected protease fragments in stimulated cells (<ref>4.10.2.</ref>). Luciferase in unstimulated cells remained
+
−
                                     inactive even at the highest amount of transfected protease fragments, proving low leakage and high inducibility of the split protease system in response to rapamycin.
+
 
                                 </p>
 
                                 </p>
 
                                 <div style="float:left; width:100%">
 
                                 <div style="float:left; width:100%">
−
                                     <figure data-ref="4.10.2.">
+
                                     <figure data-ref="4">
−
                                         <img src="https://static.igem.org/mediawiki/2016/7/72/T--Slovenia--4.10.2.png">
+
                                         <img src="https://static.igem.org/mediawiki/2016/7/76/T--Slovenia--4.9.2.png">
−
                                         <figcaption><b> Activitiy of split proteases based on rapamycin inducible system.</b>
+
                                         <figcaption><b>CRY2PHR/CIBN light inducible receptor linked with split
−
                                             <p style="text-align:justify">HEK293T cells were trasnfected with indicated cycLuc reporters and
+
                                            luciferase repeatedly responded to light.</b><br/>
−
                                                 rapamycin inducible split proteases. Whole proteases were used as positive control. An increase in luciferase activiy was detected in cells induced with
+
                                             <p style="text-align:justify">(A) Response to light depended on
−
                                                 rapamycin.
+
                                                concentration of CIBN:cLuc. After induction the cells transfected with
 +
                                                 CIBN:cLuc and CRY2PHR:nLuc were lysed and bioluminescence was measured
 +
                                                with dual luciferase assay.
 +
                                                (B) CRY2PHR light reporter was induced repeatedly. Following the
 +
                                                addition of luciferin to the medium of the cells transfected with
 +
                                                 CIBN:cLuc and CRY2PHR:nLuc
 +
                                                (ratio 1:3) were induced and left in the dark for indicated periods.
 
                                             </p>
 
                                             </p>
 
                                         </figcaption>
 
                                         </figcaption>
 
                                     </figure>
 
                                     </figure>
 
                                 </div>
 
                                 </div>
−
                                 <div style="float:right; width:50%">
+
                                <p>As the measured signal was the highest with a 1:3 ratio of CRY2PHR/CIBN (
−
                                     <figure data-ref="4.10.3.">
+
                                    <ref>4</ref>
−
                                         <img src="https://static.igem.org/mediawiki/2016/a/ac/T--Slovenia--4.10.3.png">
+
                                    A), all subsequent experiments were performed with this ratio. Next we tested if
−
                                         <figcaption><b>Kinetics of split PPVp induction with rapamycin.</b>
+
                                    this system could be induced repeatedly in real time. The CRY2PHR/CIBN system shows
−
                                             <p style="text-align:justify">HEK293T cells were trasnfected with cycLuc_PPVs and rapamycin induclible split PPVp or
+
                                    maximum activity after 2 minutes of induction, returns to background in 10 minutes
−
                                                 whole PPVp. Cells were induced with rapamycin, luciferase activity was measured in cells lysed at indicated time points.
+
                                    after
 +
                                    the stimulus is removed, and can be induced repeatedly (
 +
                                    <ref>4</ref>
 +
                                    B).
 +
                                </p>
 +
 
 +
                                 <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">
 +
                                         <figcaption><b>Testing CRY2PHR/CIBN light-inducible system with split
 +
                                            protease.</b><br/>
 +
                                             <p style="text-align:justify">(A) Schematic representation of CRY2PHR/CINB
 +
                                                light-inducible system with split protease linked to cyclic luciferase
 +
                                                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>
 
                                     </figure>
 
                                     </figure>
 
                                 </div>
 
                                 </div>
−
                                 <p>Additionally, we tested the kinetics of rapamycin induction with PPVp and its corresponding cycLuc reporter. We showed that luciferase activity starts increasing just a
+
                            </div>
−
                                     few minutes after the addition of rapamycin, resulting in activity comparable to activity of the reporter in the presence of constitutively active whole protease within one
+
                            <div>
−
                                     hour after the induction (<ref>4.10.3.</ref>).</p>
+
                                <h3 style="clear:both;"><span id="lig" class="section">&nbsp;</span>Light inducible
 +
                                    protease</h3>
 +
                                 <p style="clear:right">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) (
 +
                                    <ref>5</ref>
 +
                                    A) and tested their activity and orthogonality with bioluminescence assays (
 +
                                     <ref>5</ref>
 +
                                    B) and
 +
                                    western blotting (
 +
                                    <ref>5</ref>
 +
                                    C). The bioluminescence assay was based on the <a
 +
                                            href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Reporters">split
 +
                                        firefly luciferase</a>
 +
                                    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.
 +
                                </p>
  
−
                                 <p>After demonstrating that the new proteases are active as split enzymes with rapamycin-induced complementation, we adapted the same split system to other inputs. To
+
                                 <div style="float:left; width:100%">
−
                                     connect the split protease-based signaling to mechanosensing, we prepared <a href="https://2016.igem.org/Team:Slovenia/Mechanosensing/CaDependent_mediator"> Ca<sup>2+</sup> inducible
+
                                     <figure data-ref="6">
−
                                        proteases </a> based on calmodulin and M13 interaction. The first results look promising; however we have to confirm them in the repeated experiments. As an additional type
+
                                        <img id="cleavable luciferase"
−
                                    of input we prepared <a href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Light_dependent_mediator">light inducible split proteases</a>.</p>
+
                                            src="https://static.igem.org/mediawiki/2016/4/44/T--Slovenia--4.9.4.png">
−
                                 <p style="clear:both"> </p>
+
                                        <figcaption><b>Detection of a substrate cleavage by CRY2PHR/CIBN split protease
 +
                                            induced by light.</b><br/>
 +
                                            <p style="text-align:justify">(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.
 +
                                            </p>
 +
                                        </figcaption>
 +
                                    </figure>
 +
                                </div>
 +
                                <p style="clear:both">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 (
 +
                                    <ref>6</ref>
 +
                                    ). We showed that substrates carrying specific target peptide for proteases were
 +
                                    cleaved after
 +
                                    induction of split protease reporters.
 +
                                </p>
 +
                                 <p style="clear:left">Both methods showed a successful, fast and dose-dependent
 +
                                    response. <b>This is the first time split TEV protease has been shown to work as a
 +
                                        light inducible system.</b>
 +
                                    Also this is the first time TEVpE and PPVp were prepared as split proteins and shown
 +
                                    to function in an inducible system.
 +
                                </p>
 
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Revision as of 21:54, 18 October 2016

Light dependent mediator

Split proteases Achievements Motivation LOVpep & ePDZb CRY2-CIB1 Light inducible
protease Logic

nbsp;Light-dependent input signal and proteases

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

 

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.

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 Phy/PIF 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 . 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 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. As LOV2 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.

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 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 B).

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 ( 2 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.

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.

 CRY2-CIB1

As it has previously been shown on the example of split Cre recombinase Kennedy2010 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: 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 linked with split luciferase repeatedly responded to light.

(A) Response to light depended on concentration of CIBN:cLuc. After induction the cells transfected with CIBN:cLuc and CRY2PHR:nLuc were lysed and bioluminescence was measured with dual luciferase assay. (B) CRY2PHR light reporter was induced repeatedly. Following the addition of luciferin to the medium of the cells transfected with CIBN:cLuc and CRY2PHR:nLuc (ratio 1:3) were induced and left in the dark for indicated periods.

As the measured signal was the highest with a 1:3 ratio of CRY2PHR/CIBN ( 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 shows maximum activity after 2 minutes of induction, returns to background in 10 minutes after the stimulus is removed, and can be induced repeatedly ( 4 B).

Testing CRY2PHR/CIBN light-inducible system with split protease.

(A) Schematic representation of CRY2PHR/CINB light-inducible system with split protease linked to cyclic luciferase 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.

 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