Difference between revisions of "Team:Slovenia/Description"

(Undo revision 384800 by Zigapusnik (talk))
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     <title>Protease inducible secretion</title>
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     <title>Description</title>
 
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                 </a>
 
                 </a>
 
                 <div class="ui vertical sticky text menu">
 
                 <div class="ui vertical sticky text menu">
−
                     <a class="item" href="//2016.igem.org/Team:Slovenia/CoiledCoilInteraction">
+
                     <a class="item" href="//2016.igem.org/Team:Slovenia" style="color:#DB2828;">
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                         <i class="chevron circle left icon"></i>
+
                         <i class="selected radio icon"></i>
−
                         <b>CC interaction model</b>
+
                         <b>Home</b>
 
                     </a>
 
                     </a>
−
                  <a class="item" href="//2016.igem.org/Team:Slovenia/Demonstrate" style="color:#DB2828;">
+
                    <a class="item" href="#intro" style="margin-left: 10%">
 
                         <i class="selected radio icon"></i>
 
                         <i class="selected radio icon"></i>
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                         <b>Protease-based</b> <br />
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                         <b>Project</b>
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<b style="margin-left: 12%">inducible secretion</b>
+
 
                     </a>
 
                     </a>
−
                     <a class="item" href="#ach" style="margin-left: 10%">
+
                     <a class="item" href="#experts" style="margin-left: 10%">
 +
                        <i class="selected radio icon"></i>
 +
                        <b>Abstract for experts</b>
 +
                    </a>
 +
                    <a class="item" href="#plain" style="margin-left: 10%">
 +
                        <i class="selected radio icon"></i>
 +
                        <b>Abstract in plain English</b>
 +
                    </a>
 +
                    <a class="item" href="#achievements" style="margin-left: 10%">
 
                         <i class="selected radio icon"></i>
 
                         <i class="selected radio icon"></i>
 
                         <b>Achievements</b>
 
                         <b>Achievements</b>
 
                     </a>
 
                     </a>
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                     <a class="item" href="#intro" style="margin-left: 10%">
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                     <a class="item" href="#requirements" style="margin-left: 10%">
 
                         <i class="selected radio icon"></i>
 
                         <i class="selected radio icon"></i>
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                         <b>Introduction</b>
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                         <b>Medal requirements</b>
 
                     </a>
 
                     </a>
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                    <a class="item" href="#lum" style="margin-left: 10%">
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<a class="item" href="#imp" style="margin-left: 10%">
 
                         <i class="selected radio icon"></i>
 
                         <i class="selected radio icon"></i>
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                         <b>ER lumen</b>
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                         <b>Part improvement</b>
 
                     </a>
 
                     </a>
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                    <a class="item" href="#mem" style="margin-left: 10%">
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                     <a class="item" href="//2016.igem.org/Team:Slovenia/Idea/Challenge">
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                        <i class="selected radio icon"></i>
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                        <b>ER membrane</b>
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                    </a>
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                     <a class="item" href="//2016.igem.org/Team:Slovenia/Proof">
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                         <i class="chevron circle right icon"></i>
 
                         <i class="chevron circle right icon"></i>
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                         <b>Touch painting</b>
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                         <b>Idea</b>
 
                     </a>
 
                     </a>
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<span>
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                    <span>
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<br />
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<br/>
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</span>
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</span>
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<a href = "//2016.igem.org/Team:Slovenia/Proof"><img onmouseenter="tooglePopup();" onmouseleave="tooglePopup();" class="ui large circular image" src="//2016.igem.org/wiki/images/6/67/T--Slovenia--iGEM-gif.gif"></a>
+
                    <img onmouseenter="tooglePopup();" onmouseleave="tooglePopup();" class="ui large circular image" src="//2016.igem.org/wiki/images/c/cb/T--Slovenia--igemLogo.gif">
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                 </div>
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                    <!-- <p style="font-size:11px;">
 +
                            Touchpaint - detecting touch by light. The iGEM symbol was drawn with a glass rod letter by letter on engineered human cells and imaged by a camera.
 +
                            Cells were transfected with constructs coding for the bacterial ion channel MscS, gas vesicles (GvpA and GvpC) to enhance mechanosensing and a Ca-dependent
 +
                            cyclic split luciferase reporter to visualize the signal by light.
 +
                    </p> -->
 +
                    <div class="popup igemSign">
 +
<span class="popuptext" id="igemSign">Touchpaint - detecting touch by light. The iGEM symbol was drawn with a glass rod letter by letter on engineered human cells and imaged by a camera.
 +
Cells were transfected with constructs coding for the bacterial ion channel MscS, gas vesicles (GvpA and GvpC) to enhance mechanosensing and a Ca-dependent
 +
                            cyclic split luciferase reporter to visualize the signal by light.</span>
 +
                    </div>
 +
                 </div>  
  
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             </div>
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             </div>  
 
             <div class="article" id="context">
 
             <div class="article" id="context">
 
                 <!-- menu goes here -->
 
                 <!-- menu goes here -->
 
                 <!-- content goes here -->
 
                 <!-- content goes here -->
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                 <div class="main ui citing justified container">
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                    <div class="main ui citing justified container">
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                    <div>
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<div>
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                        <h1 class="ui centered dividing header"><span id="intro" class="section colorize"> &nbsp; </span>Sonicell
−
<h1 class="ui left dividing header"><span id="ach" class="section colorize">&nbsp;</span>Protease-based inducible
+
                        </h1>
−
secretion</h1>
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−
 
+
−
<div class="ui segment" style="background-color: #ebc7c7;">
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−
+
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<p><b>
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<ul>
+
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<li>Retention of proteins in ER lumen was demonstrated by confocal microscopy and
+
−
detection of the protein in the cell medium.
+
−
<li>A variant of TEVp active in the ER lumen was implemented to control protein
+
−
secretion from the ER lumen.
+
−
<li>Retention of proteins on the ER membrane was also demonstrated by confocal
+
−
microscopy and detection of the protein in the cell medium.
+
−
<li>Rapamycin induced cleavage was used for controlled and inducible secretion of
+
−
proteins from the ER membrane.
+
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</ul>
+
−
</b></p>
+
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</div>
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                        </div>
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                         <div class="ui segment">
 
                         <div class="ui segment">
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<div>
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                             <p>Project Sonicell introduces exciting foundational advances to synthetic biology aimed to
−
<h4><span id = "intro" class="section colorize">&nbsp;</span></h4>
+
                                enable rapid cellular response to a combination of external stimuli such as sound, light
−
                             <p>To achieve a fast regulated cellular response resulting in the protein release, we
+
                                 or chemical compounds. This system is composed of a module for <b>enhanced sensitivity of
−
                                 decided to mimic the release of insulin from beta cells where the protein of
+
                                 cells to ultrasound or other mechanical stimuli</b>, sensed by a calcium-dependent reporter,
−
                                 interest is pre-formed and present intracellularly in secretory granules. In contrast to the
+
                                 and a module for integration of a combination of several input signals into a <b>signaling
−
                                 specialized storage and release mechanism of insulin from beta cells we wanted to
+
                                 pathway based on proteolysis </b> by a collection of orthogonal proteases. Finally, the proteases were
−
                                develop a more general and modular solution by making use of components already existing
+
                                 designed to cleave an endoplasmic reticulum retention signal from target proteins, which
−
                                 in different types of cells. Additionally, there should be minimal leakage from the
+
                                 results in <b>secretion of premade proteins</b>.</p>
−
                                 protein depot in the uninduced state and after induction secretion from the cell should
+
                             <div>
−
                                 be fast.</p>
+
                                <div class="container">
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+
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                             <div class="ui styled fluid accordion">
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                                        style="border-radius: 15px;"
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                                <div class="title">
+
                                        src="//2016.igem.org/wiki/images/c/c2/T--Slovenia--Main-scheme0.png"
−
                                     <i class="dropdown icon"></i>
+
                                        alt="project scheme" usemap="#projectmap" id="projectScheme"/>
−
                                    Further explanation ...
+
                                     <map name="projectmap">
−
                                </div>
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                                        <area id="area1" class="popup" shape="poly" coords="" alt="module1"
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                                <div class="content">
+
                                              onmouseover="loadImage('//2016.igem.org/wiki/images/9/92/T--Slovenia--Main-scheme1.png', 'module1')"
−
                                    <p> Not many systems for the inducible release of proteins have been engineered to
+
                                              onmouseout="loadImage('//2016.igem.org/wiki/images/c/c2/T--Slovenia--Main-scheme0.png', 'module1')"
−
                                        date. In one of the few examples Rivera et al. developed a system where the
+
                                              href="//2016.igem.org/Team:Slovenia/Mechanosensing/Overview">
−
                                        protein of interest
+
                                        <area id="area2" class="popup" shape="poly" coords="" alt="module1"
−
                                        was fused to a conditional aggregation domain (CAD)
+
                                              onmouseover="loadImage('//2016.igem.org/wiki/images/5/54/T--Slovenia--Main-scheme3.png', 'module2')"
−
                                         <x-ref>Rivera2000</x-ref>
+
                                              onmouseout="loadImage('//2016.igem.org/wiki/images/c/c2/T--Slovenia--Main-scheme0.png', 'module2')"
−
                                        . These domains form aggregates in the endoplasmic reticulum (ER) that are too
+
                                              href="//2016.igem.org/Team:Slovenia/Protease_signaling/Logic">
−
                                        large to exit the ER.
+
                                        <area id="area3" class="popup" shape="poly" coords="" alt="module1"
−
                                        After the addition of a small synthetic molecule, the CADs start to disaggregate
+
                                              onmouseover="loadImage('//2016.igem.org/wiki/images/f/f6/T--Slovenia--Main-scheme2.png', 'module3')"
−
                                        and the protein of interest can be secreted. In the second example Chen et al.
+
                                              onmouseout="loadImage('//2016.igem.org/wiki/images/c/c2/T--Slovenia--Main-scheme0.png', 'module3')"
−
                                        introduced a
+
                                              href="//2016.igem.org/Team:Slovenia/Protease_signaling/Overview">
−
                                         light-triggered secretion system. They also based their system on conditional
+
                                        <area id="area4" class="popup" shape="poly" coords="" alt="module1"
−
                                        aggregation; however they used the plant photoreceptor UVR8 which forms
+
                                              onmouseover="loadImage('//2016.igem.org/wiki/images/1/12/T--Slovenia--Main-scheme4.png', 'module4')"
−
                                        photolabile homodimers to make
+
                                              onmouseout="loadImage('//2016.igem.org/wiki/images/c/c2/T--Slovenia--Main-scheme0.png', 'module4')"
−
                                        aggregates on the ER membrane. Upon light excitation the aggregates made by UVR8
+
                                              href="//2016.igem.org/Team:Slovenia/Implementation/ProteaseInducible_secretion">
−
                                        started to disaggregate and were transported from the ER to the plasma membrane,
+
                                    </map>
−
                                        but have not been
+
                                    <div class="popup module1">
−
                                         observed in the cell supernatant
+
                                        <span class="popuptext" id="module1"><b>Enhanced mechanosensing:</b><br>
−
                                        <x-ref>Chen2013</x-ref>.
+
                                            Sensitivity of mammalian cells to ultrasound or other mechanical stimuli was enhanced by the introduction
−
                                        The weakness of the two described systems is that they both rely on the
+
                                            of mechanosensitive ion channels and/or by the expression of protein gas vesicles from bacteria. Influx of
−
                                        exogenous chemical or physical signals instead of using a biochemical signal to
+
                                            calcium through channels is sensed by formation of a complex between calmodulin and M13 peptide that can
−
                                        induce the secretion, which
+
                                            result in a rapid light emission by cells (used for cell painting) or reconstitution of a split protease.</span>
−
                                        means that they can’t be integrated into the signaling system that’s senses the
+
                                    </div>
−
                                        cellular state. In order to better respond to the state of the cell or a logic
+
                                    <div class="popup module2">
−
                                        circuit inside a cell
+
                                         <span class="popuptext" id="module2"><b>Protease based signaling <br/>and information processing:</b><br/>
−
                                        we decided to develop an inducible secretion system based on the biochemical
+
                                            Combinations of proteolytic activities against specific targets resulted in activation of a reporter or another
−
                                        signal.
+
                                            protease, which forms the basis for the design of a new type of rapid signaling pathways and construction of
−
                                     </p>
+
                                            logic functions.</span>
 +
                                    </div>
 +
                                    <div class="popup module3">
 +
                                         <span class="popuptext" id="module3"><b>Orthogonal site-specific proteases: </b><br/> A collection of orthogonal
 +
                                            site-specific proteases that recognize different targets was prepared as split proteins, whose activity against
 +
                                            selected targets can be induced by stimulation with an external signal such as light or chemicals.</span>
 +
                                    </div>
 +
                                    <div class="popup module4">
 +
                                         <span class="popuptext"
 +
                                              id="module4"> <b>Protease-triggered rapid secretion <br/> of therapeutic proteins:</b> <br/>A rapid cellular
 +
                                            response by secretion of a protein is triggered by the proteolytic cleavage of an endoplasmic reticulum retention
 +
                                            peptide. After the cleavage the cargo protein is moved from the ER, and secreted as therapeutic protein.</span>
 +
                                     </div>
 
                                 </div>
 
                                 </div>
 
                             </div>
 
                             </div>
−
                            <p><br/>Many proteins that reside on the membrane or in the lumen of the ER contain short
 
−
                                peptide signals. Proteins present in the lumen of the ER contain a KDEL C-terminal
 
−
                                sequence
 
−
                                (Lys-Asp-Glu-Leu) while type I transmembrane (TM) proteins contain a dilysine (KKXX)
 
−
                                motif on their C-terminus (cytosolic side)
 
−
                                <x-ref>Munro1987, Jackson1990, Stornaiuolo2003</x-ref>.
 
−
                                . The mechanism that allows these proteins to stay in the ER is retrieval rather than
 
−
                                retention. However, we decided to use the term retention for
 
−
                                description of this process. ER luminal proteins interact with the KDEL receptor, a
 
−
                                transmembrane ER resident protein. The cytosolic part of the KDEL receptor interacts
 
−
                                with
 
−
                                coat proteins I (COP I) which coat vesicles and are responsible for transporting
 
−
                                proteins from the cis end of the Golgi apparatus (cis-GA) back to the ER. The KKXX motif
 
−
                                present
 
−
                                on type I TM proteins can directly interact with the COP I for retrieval
 
−
                                <x-ref>Stornaiuolo2003, Letourneur1994</x-ref>
 
−
                                .
 
−
                            </p>
 
−
                            <p>Our idea was that if we proteolytically remove the retention signal, the protein of
 
−
                                interest would no longer be retrieved back to the ER and could be secreted from the
 
−
                                cell.
 
−
                                To achieve this we designed two types of secretory reporters, one type based on the
 
−
                                luminal retention using KDEL sequence and the other based on the transmembrane retention
 
−
                                with
 
−
                                a KKMP sequence. In each case, the retention sequence was preceded by a TEVp cleavage
 
−
                                site to allow for inducible secretion, which could be replaced by any other peptide
 
−
                                target
 
−
                                of our <a href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Orthogonality"> orthogonal protease set</a>.</p>
 
 
                         </div>
 
                         </div>
−
                         </div>
+
                    </div>
−
 
+
                    <div class="ui segment">
−
                         <h1><span class="section">&nbsp;</span>Results</h1>
+
                         <h4><span id="experts" class="section colorize">&nbsp;</span>Abstract for experts</h4>
 +
                        <p>Synthetic biology opens exciting perspectives to control cells, for applications ranging from
 +
                            industrial processes to cell-based therapy. However, the large majority of designed cellular
 +
                            circuits are based on transcriptional regulation, which may be too slow for many
 +
                            therapeutic or diagnostic applications, for example delivery of insulin or detection of a
 +
                            metabolite. Several medical doctors and researchers that we consulted stressed that a fast
 +
                            but controllable response is high on their wish list of expectations from synthetic biology.
 +
                            Additionally, noninvasive stimulation of selected tissues in the organism would also be
 +
                            highly desirable. While light is extremely useful as a rapid, spatially-restricted input
 +
                            signal, it cannot penetrate deep into the tissue. On the other hand, ultrasound combines
 +
                            several advantages of light with the added ability to penetrate tissue.</p>
 +
                        <p>In our project we enhanced the sensitivity of mammalian cells to ultrasound or other
 +
                            mechanical stimuli by introduction of bacterial or engineered mammalian mechanosensors.
 +
                            Additionally, the response to ultrasound and touch was strongly increased by expression of
 +
                            the two components of bacterial gas vesicles, GvpA and GvpC. Mechanosensing was detected by
 +
                            the calcium-induced calmodulin-M13 complex reconstituting split cyclic luciferase, highly
 +
                            applicable for the emerging field of mechanogenetics. This enabled us to draw on cells using
 +
                            touch, where we engaged in collaboration with the artist Laura Olalde.</p>
 +
                        <p>For the rapid response of cells to multiple stimuli we designed proteolysis-based signaling
 +
                            pathways. Four orthogonal split proteases were generated, each recognizing
 +
                            its own motif of seven amino acid residues. Based on cleavage of coiled-coil dimerizing
 +
                            domains we demonstrated the ability to implement proteolysis-based signal pathways and logic
 +
                            functions in mammalian cells. Based on the cleavage of an ER retention peptide by a
 +
                            protease, input signals led to protein secretion without the slow step of induced protein
 +
                            synthesis.</p>
 +
                        <p>We believe that this project introduced several foundational advances that could be very
 +
                            useful to synthetic biology far beyond iGEM and for the benefit of humanity for therapy,
 +
                            diagnostics and potentially many other advanced applications.</p>
 +
                    </div>
 +
                    <div class="ui segment">
 +
                        <h4><span id="plain" class="section colorize">&nbsp;</span>Abstract in plain English</h4>
 +
                        <p>
 +
                            Synthetic biology aims to control cells so they can obey our commands and do what we want,
 +
                            for example produce drugs when needed. In our project we made cells which respond to ultrasound
 +
                            or touch. When we touch the cells they light up, which can be recorded on a camera. Ideally
 +
                            we want cells to respond to our commands as fast as possible, because sometimes we can’t
 +
                            wait an hour before the cells produce the medicine and release it. That is why we gave cells
 +
                            a novel mechanism of processing information.
 +
                            We achieved this by combining several enzymes that recognize very specific parts of proteins
 +
                            and cut them, which changes their function. This allowed us to combine different signals,
 +
                            like sound, touch, light or chemicals, to obtain the desired cell response. The new enzymes
 +
                            can also cut the anchor with which medicines are attached to cells after the cells make
 +
                            them. Among many possible uses of our inventions, we can imagine activating cells in the
 +
                            brain by ultrasound, which means that we don’t need to use
 +
                            surgery to help people with Parkinson’s disease, or can trigger fast production of insulin
 +
                            in the body, to help people with diabetes.
 +
                        </p>
 +
                    </div>
 +
                    <div>
 +
                         <h1 class="ui centered dividing header"><span id="achievements" class="section colorize"> &nbsp; </span>Achievements
 +
                        </h1>
 
                         <div class="ui segment">
 
                         <div class="ui segment">
−
<div>
+
                            <div class="corners" style="float:right;">
−
                            <h3><span id = "lum" class="section colorize">&nbsp;</span>Secretion from the ER lumen</h3>
+
                                <p><img src="//2016.igem.org/wiki/images/d/dc/T--Slovenia--starSmall.png"
−
                            <p>To achieve and detect the inducible secretion from the ER lumen, we created two reporter
+
                                        alt="newAtiGEM" width="28" height="28" style="display: inline;"> new in science
−
                                constructs with a cleavable KDEL sequence targeted to the ER lumen: SEAP<sup>KDEL</sup>
+
                                 </p>
−
                                and
+
                                 <p><img src="//2016.igem.org/wiki/images/0/0d/T--Slovenia--igemLogoSmall.png"
−
                                TagRFP<sup>KDEL</sup>. Those proteins contained a protease target motif between the
+
                                         alt="newAtiGEM" width="30" height="30" style="display: inline;"> new at iGEM</p>
−
                                reporter domain and the KDEL domain, aimed to enable protein secretion after the
+
−
                                proteolytic cleavage.
+
−
                                We used a TEVp variant (erTEVp) for all of our experiments with luminal retention.</p>
+
−
                            <div class="ui styled fluid accordion">
+
−
                                <div class="title">
+
−
                                    <i class="dropdown icon"></i>
+
−
                                    Further explanation ...
+
−
                                 </div>
+
−
                                 <div class="content">
+
−
                                    <p>
+
−
                                        In order to rely on TEVp cleavage in the ER lumen, we had to take some
+
−
                                        additional considerations into account. Cesaratto et al.</x-ref>
+
−
                                        Cesaratto2015</x-ref> reported that the wild
+
−
                                        type TEV protease is not active in the lumen of ER. They designed a TEV protease
+
−
                                        variant active in the endoplasmic reticulum by preventing two major types of
+
−
                                        post-translational
+
−
                                        modifications: N-glycosylation and cysteine oxidation. To avoid these inhibiting
+
−
                                         modifications, mutations N23Q, C130S and N171T were made. To ensure correct
+
−
                                        localization and
+
−
                                        accumulation of this TEVp variant inside the endoplasmic reticulum, we also
+
−
                                        attached an ER signal sequence at the N-terminus and KDEL at the C-terminus of the
+
−
                                        protein.
+
−
                                    </p>
+
−
                                </div>
+
 
                             </div>
 
                             </div>
−
                             <p style="clear:both"><br/></p>
+
                             <ul>
−
 
+
                                 <li>Mammalian cell sensitivity to ultrasound and mechanical stimuli was increased by
−
 
+
                                     ectopic expression of bacterial or human cation permeable channels and functional
−
                            <div style="float:right; width:50%">
+
                                    reconstitution of bacterial protein gas vesicles from two protein components (GvpA
−
                                 <figure data-ref="1">                                  
+
                                    and GvpC) <img src="//2016.igem.org/wiki/images/d/dc/T--Slovenia--starSmall.png"
−
                                     <img src="https://static.igem.org/mediawiki/2016/9/9e/T--Slovenia--6.2.1.png">
+
                                                  alt="newAtiGEM" width="28" height="28" style="display: inline;"></li>
−
                                    <figcaption><b>Cleavage with ER-residing protease (erTEV) facilitates secretion of
+
                                 <li>A custom-made ultrasound generator device was used to stimulate mammalian cells <img
−
                                        reporter from cells.</b><br/>
+
                                        src="//2016.igem.org/wiki/images/0/0d/T--Slovenia--igemLogoSmall.png"
−
                                        <p style="text-align:justify">(A) Scheme of the reporter with cleavable KDEL
+
                                         alt="newAtiGEM" width="30" height="30" style="display: inline;"></li>
−
                                            retention signal and protease target motif.
+
                                <li>A mechano-sensory luciferase reporter sensitive to an influx of free calcium ions
−
                                            (B) The reporter with the KDEL retention signal was localized in the ER.
+
                                    was introduced into mammalian cells, which enabled rapid light emission of mammalian
−
                                            HEK293T cells were transfected with the indicated reporters and in (C) also
+
                                    cells in response to mechanical stimuli and enabled painting on cells by touch with
−
                                            with erTEVp.
+
                                    exciting potentials for other applications <img
−
                                            Localization was detected with confocal microscopy. (C)The reporter was
+
                                            src="//2016.igem.org/wiki/images/d/dc/T--Slovenia--starSmall.png"
−
                                            detected in the medium of cells only when cotransfected with erTEVp. HEK293T
+
                                            alt="newAtiGEM" width="28" height="28" style="display: inline;"></li>
−
                                            cells were transfected
+
                                 <li>A cyclic proteolysis-activated luciferase reporter was experimentally verified and
−
                                            with the indicated constructs. Reporters were detected with WB in the
+
                                     introduced into the iGEM collection <img
−
                                            concentrated medium.
+
                                            src="//2016.igem.org/wiki/images/0/0d/T--Slovenia--igemLogoSmall.png"
−
                                        </p></figcaption>
+
                                            alt="newAtiGEM" width="30" height="30" style="display: inline;"></li>
−
                                </figure>
+
                                <li>A set of four different orthogonal site-specific proteases was designed and tested
−
                            </div>
+
                                    as split proteins with induced reconstitution in mammalian cells <img
−
                            <p>When the TagRFP<sup>KDEL</sup> reporter (
+
                                             src="//2016.igem.org/wiki/images/d/dc/T--Slovenia--starSmall.png"
−
                                <ref>1</ref>
+
                                            alt="newAtiGEM" width="28" height="28" style="display: inline;"></li>
−
                                A) was expressed in the ER without an active erTEVp we confirmed its localization in the
+
                                 <li>New orthogonal protease-based signaling pathways were designed as an information      processing platform
−
                                ER with confocal microscopy
+
                                  and several logic functions based on the combination of multiple input
−
                                (
+
                                     signals were tested experimentally <img
−
                                <ref>1</ref>
+
                                            src="//2016.igem.org/wiki/images/d/dc/T--Slovenia--starSmall.png"
−
                                B). Additionally, we could not detect any TagRFP in the cell medium with Western
+
                                            alt="newAtiGEM" width="28" height="28" style="display: inline;"></li>
−
                                blotting. When erTEVp was present and active in the ER, the KDEL sequence was
+
                                 <li>Proteolysis of ER retention signal was introduced as the trigger for the fast
−
                                removed from the reporter and the protein was secreted from the cell, which we detected
+
                                    release of proteins from cells aimed to enable fast therapeutic responses such as
−
                                with Western blot (
+
                                    required for the release of peptide hormones, neuroactive peptides etc. <img
−
                                <ref>1</ref>
+
                                            src="//2016.igem.org/wiki/images/d/dc/T--Slovenia--starSmall.png"
−
                                C). This demonstrated that proteolytic activity in the ER can
+
                                            alt="newAtiGEM" width="28" height="28" style="display: inline;"></li>
−
                                regulate protein secretion.
+
                             </ul>
−
                            </p>
+
−
                            <p style="clear:left">Using SEAP<sup>KDEL</sup> we were able to confirm that the reporter is
+
−
                                not present in the cell medium without coexpression of erTEVp. When erTEVp was
+
−
                                cotransfected with
+
−
                                the reporter, we detected a large increase in enzymatic activity in the medium (
+
−
                                 <ref>2</ref>
+
−
                                ).
+
−
                            </p>
+
−
                            <div style="float:left; width:40%">
+
−
                                <figure data-ref="2">
+
−
                                    <img src="https://static.igem.org/mediawiki/2016/6/61/T--Slovenia--6.2.2.png">
+
−
                                    <figcaption><b>Secretion of the SEAP reporter from ER lumen by cleavage with
+
−
                                         ER-resident protease.</b><br/>
+
−
                                        <p style="text-align:justify">HEK293T cells were transfected with indicated
+
−
                                            reporter and erTEVp. Increased SEAP activity was detected in the medium of
+
−
                                            cells
+
−
                                            expressing both reporter and erTEVp protease.
+
−
                                        </p>
+
−
                                    </figcaption>
+
−
                                </figure>
+
−
                            </div>
+
−
                            </div>
+
−
 
+
−
<div>
+
−
                            <h3 style="clear:both"><span id = "mem" class="section colorize">&nbsp;</span>Secretion from the ER membrane</h3>
+
−
                            <p>The second approach to regulate protein secretion from the ER by protease was to used
+
−
                                KKMP ER retention peptide linked to the transmembrane protein with a protease target
+
−
                                motif on the cytoplasmic side, N-terminally to the KKMP peptide. A transmembrane (TM)
+
−
                                domain from the B-cell receptor
+
−
                                (<a href="http://parts.igem.org/Part:BBa_K157010">Bba_K157010</a>) was used for the
+
−
                                integration of target proteins in the ER membrane. Similar as described above, two
+
−
                                reporter
+
−
                                constructs with SEAP and TagRFP (SEAP:TM<sup>KKMP</sup> and TagRFP:TM<sup>KKMP</sup>)
+
−
                                were designed and the constructs also contained a signal sequence at their N-terminus
+
−
                                and a
+
−
                                proteolytically cleavable ER retention signal at their C-terminus. In case of
+
−
                                transmembrane targeted reporters we used the KKMP retention signal preceded by 3 copies
+
−
                                of the
+
−
                                TEVp cleavage site on the cytosolic side of the membrane.</p>
+
−
                            <p>Additionally, either one or four furin cleavage sites were inserted between the protein
+
−
                                of interest on the luminal side of the ER, which enable cleavage of the reporter
+
−
                                protein from the membrane, but this could only occur after the KKMP had been removed and
+
−
                                the protein could enter the trans-GA. Furin is a native cellular endoprotease that is
+
−
                                active only in the trans-GA.</x-ref>Henrich2003</x-ref> This allowed us to design our
+
−
                                constructs so that they are cleaved off of the membrane without any modified scar
+
−
                                sequences
+
−
                                attached to them.</p>
+
−
 
+
−
 
+
−
                            <div style="float:left; width:100%"><span id="tagRFP" class="section"></span>
+
−
                                 <figure data-ref="3">
+
−
                                     <img src="https://static.igem.org/mediawiki/2016/b/b4/T--Slovenia--6.2.3.png">
+
−
                                    <figcaption><b>Localization of protease-responsive reporters on ER depending on the
+
−
                                        proteolysis. </b><br/>
+
−
                                        <p style="text-align:justify">(A) The transmembrane reporter without the KKMP
+
−
                                            retention signal was localized both on the ER and plasma membrane. (B) The
+
−
                                            transmembrane reporter with the KKMP retention
+
−
                                            signal was localized exclusively on the ER membrane. (C) After cleavage of
+
−
                                            the KKMP retention signal, the transmembrane reporter translocated to the
+
−
                                            plasma membrane. HEK293T
+
−
                                            cells were transfected with the indicated reporters and in (C) also with
+
−
                                            TEVp. Localization was detected with confocal microscopy. Each image is
+
−
                                            accompanied with a scheme of
+
−
                                            the transfected construct. (D) Glycosylated reporter was detected in the
+
−
                                            medium of cells transfected with the transmembrane reporter without the KKMP
+
−
                                             retention signal.
+
−
                                            HEK293T cells were transfected with the indicated constructs. Reporters were
+
−
                                            detected with WB in the concentrated medium. In lane 2, sample was incubated
+
−
                                            with N-glycosidase
+
−
                                            F.
+
−
                                        </p></figcaption>
+
−
                                </figure>
+
−
                            </div>
+
−
                            <div style="float:right; width:50%"><span  id="SEAP" class="section"></span>
+
−
                                 <figure data-ref="4">
+
−
                                     <img onclick="resize(this)" src="https://static.igem.org/mediawiki/2016/2/2f/T--Slovenia--6.2.4.png">
+
−
                                    <figcaption><b>Inducible secretion of reporter localized on ER membrane.</b><br/>
+
−
                                        <p style="text-align:justify">SEAP activity was increased in the medium of cells
+
−
                                            induced with rapamycin. (B) Scheme of the transmembrane reporter with
+
−
                                            cleavable KKMP retention signal and inducible protease.
+
−
                                            HEK293T cells were transfected with the indicated reporter and rapamycin
+
−
                                            inducible split proteases. Uncleaved proteases were used as positive
+
−
                                            control.</p></figcaption>
+
−
                                 </figure>
+
−
                            </div>
+
−
                            <p>Localization of the TagRFP:TM<sup>KKMP</sup> reporter was confirmed by the confocal
+
−
                                microscopy. We used a control reporter without the KKMP retention signal (TagRFP:TM)
+
−
                                which we detected both on the ER and the plasma membrane (
+
−
                                <ref>3</ref>
+
−
                                A). In case of present KKMP retention signal, the reporter was detected only on the
+
−
                                ER
+
−
                                (
+
−
                                <ref>3</ref>
+
−
                                B). When TagRFP:TM<sup>KKMP</sup> was coexpressed with TEVp, localization of the
+
−
                                reporter was similar to the localization of the positive control (TagRFP:TM)
+
−
                                on the plasma membrane and the ER (
+
−
                                <ref>3</ref>
+
−
                                C).
+
−
                            </p>
+
−
                            <p>A band with a slightly larger apparent size than the expected size of TagRFP (28 kDa) was
+
−
                                detected by Western blotting in cells transfected with TagRFP:TM. We showed that the
+
−
                                unexpected difference in size was due to glycosylation, as we detected the protein at
+
−
                                the expected size after deglycosylation of the medium sample with N-glycosidase F. We
+
−
                                were
+
−
                                unable to detect a corresponding band in the medium of cells transfected with
+
−
                                TagRFP:TM<sup>KKMP</sup> in the absence of the protease.</p>
+
−
                            <p>Together, these results confirm that localization and secretion of the protein reporter
+
−
                                with the transmembrane domain depends on the presence and proteolysis of the KKMP
+
−
                                retention signal and that proteolysis can be used to induce secretion of already
+
−
                                synthesized protein.</p>
+
−
 
+
−
 
+
−
                            <p style="clear:both">Finally, we cotransfected cells with SEAP:TM<sup>KKMP</sup> and
+
−
                                rapamycin-inducible split TEVp. We detected increased levels of the SEAP enzymatic
+
−
                                activity in the medium of
+
−
                                cells stimulated with rapamycin, which was dose dependent with respect to the amount of
+
−
                                the transfected reporter-coding plasmid (
+
−
                                <ref>4</ref>
+
−
                                ). These results confirm that
+
−
                                secretion of a target protein can be made inducible by an externally supplied signal,
+
−
                                processed through our split protease system.
+
−
                            </p>
+
−
                            <p style="clear:both">
+
−
                             </p>
+
 
                         </div>
 
                         </div>
 +
                    </div>
 +
                    <div>
 +
                        <h1 class="ui centered dividing header"><span id="requirements" class="section colorize"> &nbsp; </span>Medal
 +
                            requirements</h1>
 +
                        <div class="ui basic segment">
 +
                            <table class="ui collapsing unstackable celled table" style="box-shadow: 0 1px 2px 0 rgba(34,36,38,.15)">
 +
                                <thead class="full-width">
 +
                                <tr class="center aligned">
 +
                                    <th> Medal</th>
 +
                                    <th> Criteria</th>
 +
                                    <th> Explanation</th>
 +
                                    <th></th>
 +
                                </tr>
 +
                                </thead>
 +
                                <tbody>
 +
                                <tr>
 +
                                    <td colspan="4" class="center aligned" style="background-color: gold">GOLD</td>
 +
                                </tr>
 +
                                <tr>
 +
                                    <td>Integrated Human Practices</td>
 +
                                    <td>Expand on your silver medal activity by demonstrating how you have integrated
 +
                                        the investigated issues into
 +
                                        the design and/or execution of your project.
 +
                                    </td>
 +
                                    <td>Implementation of several <a
 +
                                            href="https://2016.igem.org/Team:Slovenia/Integrated_Practices">experts from
 +
                                        different medical fields and culturologists</a> helped us
 +
                                        improve our
 +
                                        project.
 +
                                    </td>
 +
                                    <td><i class="large green checkmark icon"></i></td>
 +
                                </tr>
 +
                                <tr>
 +
                                    <td>Improve a previous part or project
 +
                                    </td>
 +
                                    <td>Improve the function OR characterization of an existing BioBrick Part or Device
 +
                                        and enter this information
 +
                                        in the Registry.
 +
                                    </td>
 +
                                    <td>We <a href="https://2016.igem.org/Team:Slovenia/Parts">improved</a> the parts
 +
                                        <a href="http://parts.igem.org/Part:BBa_K737005:Experience">BBa_K737005</a> and <a
 +
                                                href="http://parts.igem.org/Part:BBa_K157010:Experience">BBa_K157010</a>
 +
                                        by equipping them with additional tags, expressing them
 +
                                        in human cells and further characterizing their function.
 +
                                    </td>
 +
                                    <td><i class="large green checkmark icon"></i></td>
 +
                                </tr>
 +
                                <tr>
 +
                                    <td>Proof of concept</td>
 +
                                    <td>Demonstrate a functional proof of concept of your project. </td>
 +
                                    <td>We successfully demonstrated the functionality of selected (split) proteases and
 +
                                        used them to <a
 +
                                                href="https://2016.igem.org/Team:Slovenia/Demonstrate">control
 +
                                            secretion</a> of the reporter protein from the cell.
 +
                                    </td>
 +
                                    <td><i class="large green checkmark icon"></i></td>
 +
                                </tr>
 +
                                <tr>
 +
                                    <td>Demonstrate your work</td>
 +
                                    <td>How your project works under real-world conditions.
 +
                                    </td>
 +
                                    <td>We showed that our mechanosensing constructs coexpressed in human cells allow
 +
                                        for a controlled response to
 +
                                        touch and used them for our art application, <a
 +
                                                href="//2016.igem.org/Team:Slovenia/Proof">Touch painting</a>.
 +
                                    </td>
 +
                                    <td><i class="large green checkmark icon"></i></td>
 +
                                </tr>
 +
                                <tr>
 +
                                    <td colspan="4" class="center aligned" style="background-color: silver">SILVER</td>
 +
                                </tr>
 +
                                <tr>
 +
                                    <td>Validated Part / Validated Contribution</td>
 +
                                    <td>Experimentally validate that at least one new BioBrick Part or Device of your
 +
                                        own design and construction
 +
                                        works as expected.
 +
                                    </td>
 +
                                    <td>We demonstrated the functionality of our constructs and provided experimental
 +
                                        data. We created a list of our
 +
                                        <a href="https://2016.igem.org/Team:Slovenia/Parts">favorite parts</a>
 +
                                        and detailed our experiences with them.
 +
                                    </td>
 +
                                    <td><i class="large green checkmark icon"></i></td>
 +
                                </tr>
 +
                                <tr>
 +
                                    <td>Collaboration</td>
 +
                                    <td>Convince the judges you have helped any registered iGEM team from high school, a
 +
                                        different track, another
 +
                                        university, or another institution in a significant way.
 +
                                    </td>
 +
                                    <td>During our project we had several skype meetings with other iGEM teams. We also
 +
                                        provided iGEM Team biotINK
 +
                                        from Munich with <a href="http://parts.igem.org/Part:BBa_K782063">BBa_K782063
 +
                                            created by team Slovenia 2012.</a>
 +
                                    </td>
 +
                                    <td><i class="large green checkmark icon"></i></td>
 +
                                </tr>
 +
                                <tr>
 +
                                    <td>Human Practices</td>
 +
                                    <td>Demonstrate how your team has identified, investigated, and addressed one or
 +
                                        more of issues (education,
 +
                                        public engagement, public policy issues, public perception, or other activities)
 +
                                        in the context of your
 +
                                        project.
 +
                                    </td>
 +
                                    <td>Education and transmission of interest in science is an <a
 +
                                            href="https://2016.igem.org/Team:Slovenia/HP/Gold">important part of our
 +
                                        project</a>. This
 +
                                        is why we
 +
                                        prepared several lectures for high school students and also collaborated with an
 +
                                        artist who gave our project
 +
                                        a new perspective by conveying science to public through art.
 +
                                    </td>
 +
                                    <td><i class="large green checkmark icon"></i></td>
 +
                                </tr>
 +
                                <tr>
 +
                                    <td colspan="4" class="center aligned" style="background-color: #CD7F32">BRONZE</td>
 +
                                </tr>
 +
                                <tr>
 +
                                    <td>Register and attend</td>
 +
                                    <td>Register for iGEM.</td>
 +
                                    <td>We have successfully registered.</td>
 +
                                    <td><i class="large green checkmark icon"></i></td>
 +
                                </tr>
 +
                                <tr>
 +
                                    <td>Deliverables</td>
 +
                                    <td>Meet all deliverables on the Requirements page.</td>
 +
                                    <td>We met all the listed deliverables.</td>
 +
                                    <td><i class="large green checkmark icon"></i></td>
 +
                                </tr>
 +
                                <tr>
 +
                                    <td>Attribution</td>
 +
                                    <td>Create a page on your team wiki with clear attribution of each aspect of your
 +
                                        project.
 +
                                    </td>
 +
                                    <td>We created a <a href="//2016.igem.org/Team:Slovenia/Attributions">wiki page</a> describing
 +
                                        the attributions to the
 +
                                        project.
 +
                                    </td>
 +
                                    <td><i class="large green checkmark icon"></i></td>
 +
                                </tr>
 +
                                <tr>
 +
                                    <td>Part / Contribution</td>
 +
                                    <td>Document at least one new standard BioBrick Part or Device central to your
 +
                                        project and submit this part to
 +
                                        the iGEM Registry.
 +
                                    </td>
 +
                                    <td>We documented and submitted 52 standard <a
 +
                                            href="//parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2016&group=Slovenia">BioBrick
 +
                                        parts</a>.
 +
                                    </td>
 +
                                    <td><i class="large green checkmark icon"></i></td>
 +
                                </tr>
 +
                                </tbody>
 +
                            </table>
 +
 +
<div class="ui segment" style="segment colorize"><h3><span id="imp" class="section colorize">&nbsp;</span>Part improvements</h3>
 +
<p>We improved the BioBrick <a href="http://parts.igem.org/Part:BBa_K737005:Experience">BBa_K737005</a>, deposited by team OUC-China in 2012, coding for the
 +
GvpC protein. This protein is one of two proteins that compose protein gas vesicles, which were used in our project to enhance the sensitivity of mammalian
 +
cells to mechanical stimuli. We improved the part by adding a FLAG tag, which allowed us to analyze the expression of the part form the cells via western blot and
 +
to observe the subcellular localization of the protein by confocal microscopy. We further characterized the part by expressing it in mammalian cells together with
 +
GvpA, showing that these proteins substantially increase the cell sensitivity to ultrasound. This part also plays a central role in our Mechanosensing collection.</p>
 +
<p>We also improved  <a href=" http://parts.igem.org/Part:BBa_K157010:Experience"> BBa_K157010</a>, a transmembrane domain used by 2008 iGEM Freiburg team. We used this domain as
 +
an anchor to which we attached the TagRFP protein on the N-terminal side and the ER retention signal on the C-terminal side. By western blots and confocal fluorescent
 +
microcopy we were able to show that this part can not only be used for plasma membrane localization but can also be retained in the ER with the simple addition of a
 +
4-amino-acid retention signal KKMP. Upon induced proteolytic cleavage of the retention signal by inducible split protease, the protein was translocated to the trans
 +
GA and the plasma membrane, where TagRFP was cleaved by furin and secreted into the medium.</p>
 
                         </div>
 
                         </div>
−
                        <h1 class="ui left dividing header"><span id="ref-title" class="section">&nbsp;</span>References
 
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                <a href="//igem.org/Main_Page">
 
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                    <img border="0" alt="iGEM" src="//2016.igem.org/wiki/images/8/84/T--Slovenia--logo_250x250.png"
 
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 +
</div>
 +
<div>
 +
<a href="//igem.org/Main_Page">
 +
<img border="0" alt="iGEM" src="//2016.igem.org/wiki/images/8/84/T--Slovenia--logo_250x250.png" width="5%" style = "position: fixed; bottom:0%; right:1%;">
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Revision as of 12:55, 19 October 2016

Description

Home Project Abstract for experts Abstract in plain English Achievements Medal requirements Part improvement Idea
Touchpaint - detecting touch by light. The iGEM symbol was drawn with a glass rod letter by letter on engineered human cells and imaged by a camera. Cells were transfected with constructs coding for the bacterial ion channel MscS, gas vesicles (GvpA and GvpC) to enhance mechanosensing and a Ca-dependent cyclic split luciferase reporter to visualize the signal by light.

  Sonicell

Project Sonicell introduces exciting foundational advances to synthetic biology aimed to enable rapid cellular response to a combination of external stimuli such as sound, light or chemical compounds. This system is composed of a module for enhanced sensitivity of cells to ultrasound or other mechanical stimuli, sensed by a calcium-dependent reporter, and a module for integration of a combination of several input signals into a signaling pathway based on proteolysis by a collection of orthogonal proteases. Finally, the proteases were designed to cleave an endoplasmic reticulum retention signal from target proteins, which results in secretion of premade proteins.

project scheme module1 module1 module1 module1
Enhanced mechanosensing:
Sensitivity of mammalian cells to ultrasound or other mechanical stimuli was enhanced by the introduction of mechanosensitive ion channels and/or by the expression of protein gas vesicles from bacteria. Influx of calcium through channels is sensed by formation of a complex between calmodulin and M13 peptide that can result in a rapid light emission by cells (used for cell painting) or reconstitution of a split protease.
Protease based signaling
and information processing:
Combinations of proteolytic activities against specific targets resulted in activation of a reporter or another protease, which forms the basis for the design of a new type of rapid signaling pathways and construction of logic functions.
Orthogonal site-specific proteases:
A collection of orthogonal site-specific proteases that recognize different targets was prepared as split proteins, whose activity against selected targets can be induced by stimulation with an external signal such as light or chemicals.
Protease-triggered rapid secretion
of therapeutic proteins:
A rapid cellular response by secretion of a protein is triggered by the proteolytic cleavage of an endoplasmic reticulum retention peptide. After the cleavage the cargo protein is moved from the ER, and secreted as therapeutic protein.

 Abstract for experts

Synthetic biology opens exciting perspectives to control cells, for applications ranging from industrial processes to cell-based therapy. However, the large majority of designed cellular circuits are based on transcriptional regulation, which may be too slow for many therapeutic or diagnostic applications, for example delivery of insulin or detection of a metabolite. Several medical doctors and researchers that we consulted stressed that a fast but controllable response is high on their wish list of expectations from synthetic biology. Additionally, noninvasive stimulation of selected tissues in the organism would also be highly desirable. While light is extremely useful as a rapid, spatially-restricted input signal, it cannot penetrate deep into the tissue. On the other hand, ultrasound combines several advantages of light with the added ability to penetrate tissue.

In our project we enhanced the sensitivity of mammalian cells to ultrasound or other mechanical stimuli by introduction of bacterial or engineered mammalian mechanosensors. Additionally, the response to ultrasound and touch was strongly increased by expression of the two components of bacterial gas vesicles, GvpA and GvpC. Mechanosensing was detected by the calcium-induced calmodulin-M13 complex reconstituting split cyclic luciferase, highly applicable for the emerging field of mechanogenetics. This enabled us to draw on cells using touch, where we engaged in collaboration with the artist Laura Olalde.

For the rapid response of cells to multiple stimuli we designed proteolysis-based signaling pathways. Four orthogonal split proteases were generated, each recognizing its own motif of seven amino acid residues. Based on cleavage of coiled-coil dimerizing domains we demonstrated the ability to implement proteolysis-based signal pathways and logic functions in mammalian cells. Based on the cleavage of an ER retention peptide by a protease, input signals led to protein secretion without the slow step of induced protein synthesis.

We believe that this project introduced several foundational advances that could be very useful to synthetic biology far beyond iGEM and for the benefit of humanity for therapy, diagnostics and potentially many other advanced applications.

 Abstract in plain English

Synthetic biology aims to control cells so they can obey our commands and do what we want, for example produce drugs when needed. In our project we made cells which respond to ultrasound or touch. When we touch the cells they light up, which can be recorded on a camera. Ideally we want cells to respond to our commands as fast as possible, because sometimes we can’t wait an hour before the cells produce the medicine and release it. That is why we gave cells a novel mechanism of processing information. We achieved this by combining several enzymes that recognize very specific parts of proteins and cut them, which changes their function. This allowed us to combine different signals, like sound, touch, light or chemicals, to obtain the desired cell response. The new enzymes can also cut the anchor with which medicines are attached to cells after the cells make them. Among many possible uses of our inventions, we can imagine activating cells in the brain by ultrasound, which means that we don’t need to use surgery to help people with Parkinson’s disease, or can trigger fast production of insulin in the body, to help people with diabetes.

  Achievements

newAtiGEM new in science

newAtiGEM new at iGEM

  • Mammalian cell sensitivity to ultrasound and mechanical stimuli was increased by ectopic expression of bacterial or human cation permeable channels and functional reconstitution of bacterial protein gas vesicles from two protein components (GvpA and GvpC) newAtiGEM
  • A custom-made ultrasound generator device was used to stimulate mammalian cells newAtiGEM
  • A mechano-sensory luciferase reporter sensitive to an influx of free calcium ions was introduced into mammalian cells, which enabled rapid light emission of mammalian cells in response to mechanical stimuli and enabled painting on cells by touch with exciting potentials for other applications newAtiGEM
  • A cyclic proteolysis-activated luciferase reporter was experimentally verified and introduced into the iGEM collection newAtiGEM
  • A set of four different orthogonal site-specific proteases was designed and tested as split proteins with induced reconstitution in mammalian cells newAtiGEM
  • New orthogonal protease-based signaling pathways were designed as an information processing platform and several logic functions based on the combination of multiple input signals were tested experimentally newAtiGEM
  • Proteolysis of ER retention signal was introduced as the trigger for the fast release of proteins from cells aimed to enable fast therapeutic responses such as required for the release of peptide hormones, neuroactive peptides etc. newAtiGEM

  Medal requirements

Medal Criteria Explanation
GOLD
Integrated Human Practices Expand on your silver medal activity by demonstrating how you have integrated the investigated issues into the design and/or execution of your project. Implementation of several experts from different medical fields and culturologists helped us improve our project.
Improve a previous part or project Improve the function OR characterization of an existing BioBrick Part or Device and enter this information in the Registry. We improved the parts BBa_K737005 and BBa_K157010 by equipping them with additional tags, expressing them in human cells and further characterizing their function.
Proof of concept Demonstrate a functional proof of concept of your project. We successfully demonstrated the functionality of selected (split) proteases and used them to control secretion of the reporter protein from the cell.
Demonstrate your work How your project works under real-world conditions. We showed that our mechanosensing constructs coexpressed in human cells allow for a controlled response to touch and used them for our art application, Touch painting.
SILVER
Validated Part / Validated Contribution Experimentally validate that at least one new BioBrick Part or Device of your own design and construction works as expected. We demonstrated the functionality of our constructs and provided experimental data. We created a list of our favorite parts and detailed our experiences with them.
Collaboration Convince the judges you have helped any registered iGEM team from high school, a different track, another university, or another institution in a significant way. During our project we had several skype meetings with other iGEM teams. We also provided iGEM Team biotINK from Munich with BBa_K782063 created by team Slovenia 2012.
Human Practices Demonstrate how your team has identified, investigated, and addressed one or more of issues (education, public engagement, public policy issues, public perception, or other activities) in the context of your project. Education and transmission of interest in science is an important part of our project. This is why we prepared several lectures for high school students and also collaborated with an artist who gave our project a new perspective by conveying science to public through art.
BRONZE
Register and attend Register for iGEM. We have successfully registered.
Deliverables Meet all deliverables on the Requirements page. We met all the listed deliverables.
Attribution Create a page on your team wiki with clear attribution of each aspect of your project. We created a wiki page describing the attributions to the project.
Part / Contribution Document at least one new standard BioBrick Part or Device central to your project and submit this part to the iGEM Registry. We documented and submitted 52 standard BioBrick parts.

 Part improvements

We improved the BioBrick BBa_K737005, deposited by team OUC-China in 2012, coding for the GvpC protein. This protein is one of two proteins that compose protein gas vesicles, which were used in our project to enhance the sensitivity of mammalian cells to mechanical stimuli. We improved the part by adding a FLAG tag, which allowed us to analyze the expression of the part form the cells via western blot and to observe the subcellular localization of the protein by confocal microscopy. We further characterized the part by expressing it in mammalian cells together with GvpA, showing that these proteins substantially increase the cell sensitivity to ultrasound. This part also plays a central role in our Mechanosensing collection.

We also improved BBa_K157010, a transmembrane domain used by 2008 iGEM Freiburg team. We used this domain as an anchor to which we attached the TagRFP protein on the N-terminal side and the ER retention signal on the C-terminal side. By western blots and confocal fluorescent microcopy we were able to show that this part can not only be used for plasma membrane localization but can also be retained in the ER with the simple addition of a 4-amino-acid retention signal KKMP. Upon induced proteolytic cleavage of the retention signal by inducible split protease, the protein was translocated to the trans GA and the plasma membrane, where TagRFP was cleaved by furin and secreted into the medium.