Difference between revisions of "Team:Slovenia"

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     <title>Mechanosensitive channels</title>
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                     <a class="item" href="//2016.igem.org/Team:Slovenia/Mechanosensing/Overview">
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                     <a class="item" href="//2016.igem.org/Team:Slovenia" style="color:#DB2828;">
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                         <b>Overview</b>
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                         <b>Home</b>
 
                     </a>
 
                     </a>
 
                     <a class="item" href="#intro" style="margin-left: 10%">
 
                     <a class="item" href="#intro" style="margin-left: 10%">
 
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                         <b>Achievements</b>
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                         <b>Project</b>
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                    </a>
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                    <a class="item" href="#experts" style="margin-left: 10%">
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                        <i class="selected radio icon"></i>
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                        <b>Abstract for experts</b>
 
                     </a>
 
                     </a>
                     <a class="item" href="#mot" style="margin-left: 10%">
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                     <a class="item" href="#plain" style="margin-left: 10%">
 
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                         <b>Motivation</b>
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                         <b>Abstract in plain English</b>
 
                     </a>
 
                     </a>
                     <a class="item" href="#loc" style="margin-left: 10%">
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                         <b>Localization and</b>
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                         <b>Achievements</b>
                        <br/>
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                        <b style="margin-left: 12%">Expression</b>
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                     </a>
 
                     </a>
                     <a class="item" href="#us" style="margin-left: 10%">
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                     <a class="item" href="#requirements" style="margin-left: 10%">
 
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                         <b>Ultrasound stimulation</b>
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                         <b>Medal requirements</b>
 
                     </a>
 
                     </a>
                     <a class="item" href="//2016.igem.org/Team:Slovenia/Mechanosensing/Gas_vesicles">
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                     <a class="item" href="//2016.igem.org/Team:Slovenia/Idea/Challenge">
 
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                         <i class="chevron circle right icon"></i>
                         <b>Gas Vesicles</b>
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                         <b>Idea</b>
 
                     </a>
 
                     </a>
                 </div>
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                    <span>
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<br/>
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</span>
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<div style = "position:fixed; width:14%; left:3%; bottom:3%;">
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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>
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<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>
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</div>
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</div>
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                 </div>  
  
             </div>
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             </div>  
 
             <div class="article" id="context">
 
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                 <!-- menu goes here -->
 
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                        <h1 class="ui centered dividing header" style="font-size: 3rem"><span id="intro" class="section colorize"> &nbsp; </span>Sonicell
                            <h1 class="ui left dividing header"><span id="intro" class="section">&nbsp;</span>Enhanced
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                        </h1>
                                Mechanosensitivity by overexpressed <br/>Mechanosensitive Channels</h1>
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                            <div class="ui segment" style="background-color: #ebc7c7; ">
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                                <ul>
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                                    <li><b>Ectopically expressed mechanosensitive ion channels MscS and P3:FAStm:TRPC1
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                                        were used to enhance sensitivity of mammalian cells to ultrasound
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                                        stimulation.</b>
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                                    <li><b>Membrane localization of the mechanosensitive channel TRPC1 was improved by
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                                        fusing it with a FAS transmembrane domain, which also led to increased
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                                        sensitivity to ultrasound stimulation.</b>
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                                </ul>
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                            </div>
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                        </div>
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                         <div class="ui segment">
 
                         <div class="ui segment">
                             <h4><span id="mot" class="section">&nbsp;</span></h4>
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                             <p>For the therapy of diseases such as diabetes, we require a system of drug delivery that is fast, controllable and noninvasive. Engineering cells to produce drugs in the tissue is a promising direction. However, in most genetic circuits designed by synthetic biology the response is delayed due to the need to transcripe and translate the mediators. Project Sonicell introduces exciting foundational advances to synthetic biology aimed to
                            <p>We chose to test two mechanosensitive channels, human nonspecific cation channel TRPC1
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                                enable rapid cellular response to a combination of external stimuli such as sound, light
                                 and bacterial channel MscS, previously described as important receptors involved
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                                or chemical compounds. This system is composed of a module for <b>enhanced sensitivity of
                                 in the response to mechanical stimulation in human and bacteria
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                                cells to ultrasound or other mechanical stimuli</b>, sensed by a calcium-dependent reporter,
                                <x-ref>Haswell2011, Ye2013</x-ref>
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                                and a module for integration of a combination of several input signals into a <b>signaling
                                .
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                                pathway based on proteolysis </b> by a collection of orthogonal proteases. Finally, the proteases were
                            </p>
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                                 designed to cleave an endoplasmic reticulum retention signal from target proteins, which
                             <div class="ui styled fluid accordion">
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                                 results in <b>secretion of premade proteins</b>.</p>
                                 <div class="title">
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                             <div>
                                     <i class="dropdown icon"></i>
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                                 <div class="container">
                                    Further explanation ...
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                                     <img width="100%" onresize="relativeCoords();" onload="relativeCoords();"
                                </div>
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                                <div class="content">
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                                        src="//2016.igem.org/wiki/images/c/c2/T--Slovenia--Main-scheme0.png"
                                    <p>Transient receptor potential channel 1 (TRPC1) is a human non-specific cation
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                                        alt="project scheme" usemap="#projectmap" id="projectScheme"/>
                                        channel located at the plasma membrane. It has been previously reported as
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                                    <map name="projectmap">
                                        broadly expressed in human tissues where it functions as a store-operating
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                                        <area id="area1" class="popup" shape="poly" coords="" alt="module1"
                                        calcium channel
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                                        <x-ref>Xu2001</x-ref>
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                                              onmouseout="loadImage('//2016.igem.org/wiki/images/c/c2/T--Slovenia--Main-scheme0.png', 'module1')"
                                        . It belongs to the TRP superfamily that
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                                              href="//2016.igem.org/Team:Slovenia/Mechanosensing/Overview">
                                         act as tetrameric transmembrane proteins consisting of a domain formed by six
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                                        transmembrane helices, with a pore between S5 and S6
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                                        <x-ref>Nilius2007</x-ref>
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                                              onmouseout="loadImage('//2016.igem.org/wiki/images/c/c2/T--Slovenia--Main-scheme0.png', 'module2')"
                                        .
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                                              href="//2016.igem.org/Team:Slovenia/Protease_signaling/Logic">
                                         These helices present the N- and C-termini to the cytoplasm, promoting the
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                                        formation of functional homo- or hetero-tetramers
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                                        <x-ref>Bianchi2007</x-ref>
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                                              onmouseout="loadImage('//2016.igem.org/wiki/images/c/c2/T--Slovenia--Main-scheme0.png', 'module3')"
                                        .
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                                              href="//2016.igem.org/Team:Slovenia/Protease_signaling/Overview">
                                    </p>
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                                         <figure data-ref="1">
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                                            <img onclick="resize(this);"  
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                                              href="//2016.igem.org/Team:Slovenia/Implementation/ProteaseInducible_secretion">
                                                src="https://static.igem.org/mediawiki/2016/7/7a/T--Slovenia--3.2.2.PNG">
+
                                    </map>
                                            <figcaption><b>Structure of a tetrameric homologous TRPv6 channel (<a
+
                                    <div class="popup module1">
                                                    href="http://www.rcsb.org/pdb/explore.do?structureId=5IRX">PDB
+
                                        <span class="popuptext" id="module1"><b>Enhanced mechanosensing:</b><br>
                                                5IRX</a>) presented from side and top with seen ion pore.</b>
+
                                            Sensitivity of mammalian cells to ultrasound or other mechanical stimuli was enhanced by the introduction
                                             </figcaption>
+
                                            of mechanosensitive ion channels and/or by the expression of protein gas vesicles from bacteria. Influx of
                                         </figure>
+
                                            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.</span>
 +
                                    </div>
 +
                                    <div class="popup module2">
 +
                                        <span class="popuptext" id="module2"><b>Protease based signaling <br/>and information processing:</b><br/>
 +
                                             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.</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>
                                    <p>The second channel that we selected is the bacterial mechanosensitive channel
+
                                     <div class="popup module4">
                                        MscS. Its role is to mediate turgor regulation in bacteria and it is
+
                                         <span class="popuptext"
                                        activated by changes in osmotic pressure
+
                                              id="module4"> <b>Protease-triggered rapid secretion <br/> of therapeutic proteins:</b> <br/>A rapid cellular
                                        <x-ref>Perozo2003</x-ref>
+
                                            response by secretion of a protein is triggered by the proteolytic cleavage of an endoplasmic reticulum retention
                                        . It has been previously shown that MscS is a homoheptamer, each subunit is
+
                                            peptide. After the cleavage the cargo protein is moved from the ER, and secreted as therapeutic protein.</span>
                                        31kDa in
+
                                        size and contains three transmembrane helices with the N-terminus facing the
+
                                        periplasm and the C-terminus embedded in the cytoplasm
+
                                        <x-ref>Pivetti2003</x-ref>
+
                                        .
+
                                    </p>
+
                                     <div style="clear:left; width:50%">
+
                                         <figure data-ref="2">
+
                                            <img onclick="resize(this);"
+
                                                src="https://static.igem.org/mediawiki/2016/d/df/T--Slovenia--3.2.3.PNG">
+
                                            <figcaption><b>. Crystal structure of the MscS channel (<a
+
                                                    href="http://www.rcsb.org/pdb/explore.do?structureId=5AJI">PDB
+
                                                5AJI</a>) presented from the side view with presented membrane lipids
+
                                                and from the top with seen ion pore</b></figcaption>
+
                                        </figure>
+
 
                                     </div>
 
                                     </div>
 
                                 </div>
 
                                 </div>
 
                             </div>
 
                             </div>
 
                         </div>
 
                         </div>
                         <h1><span class="section">&nbsp;</span>Results</h1>
+
                    </div>
 +
                    <div class="ui segment">
 +
                        <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;">
                                <h4><span id="loc" class="section">&nbsp;</span>Localization and expression</h4>
+
                                 <p><img src="//2016.igem.org/wiki/images/d/dc/T--Slovenia--starSmall.png"
                                <p>The mechanosensitive TRPC1 channel with each subunit comprising six transmembrane
+
                                         alt="newAtiGEM" width="28" height="28" style="display: inline;"> new in science
                                    helices (
+
                                    <ref>3</ref>
+
                                    A) and the MscS channel with three transmembrane
+
                                    helices (
+
                                    <ref>3</ref>
+
                                    A) were expressed in HEK293T cells (
+
                                    <ref>3</ref>
+
                                    D). MscS was detected as the 31 kDa band. TRPC1 was observed at 60 kDa, which was
+
                                    lower t
+
                                    han expected. We observed that the membrane localization in HEK293 was more evident
+
                                    for MscS (
+
                                    <ref>3</ref>
+
                                    B) rather than TRPC1 (
+
                                    <ref>3</ref>
+
                                    C).
+
                                </p>
+
                                <div style="clear:left; width:100%">
+
                                    <figure data-ref="3">
+
                                        <img src="https://static.igem.org/mediawiki/2016/1/1e/T--Slovenia--3.2.2.png">
+
                                        <figcaption><b>Localization and expression of mechanosensitive ion channels MscS
+
                                            and TRPC1. </b><br/>
+
                                            <p style="text-align:justify">(A) Scheme of bacterial ion channel MscS (upper) and human ion channel TRPC1
+
                                            (lower).
+
                                            (B) Ion channel MscS localized to plasma membrane. (C) TRPC1 predominantly
+
                                            localized in the ER.
+
                                            (D) Ion channels MscS and TRPC1 were expressed in HEK293 cells. HEK293 cells
+
                                            were transfected with plasmids encoding HA tagged MscS or Myc-tagged TRPC1.
+
                                            Expression by Western blot and localization by confocal microscopy were
+
                                            analyzed using anti-HA and anti-Myc antibodies, respectively.
+
</p>
+
</figcaption>
+
                                    </figure>
+
                                </div>
+
 
+
                                <p style="clear:both">To improve membrane localization of TRPC1 we fused a FAS
+
                                    transmembrane domain to TRPC1 (
+
                                    <ref>4</ref>
+
                                    A), since the
+
                                    transmembrane FAS domain has been very efficient in Jerala lab for the membrane
+
                                    localization
+
                                    <x-ref>Majerle2015</x-ref>
+
                                    . The strategy of adding an
+
                                    additional transmembrane domain has to own knowledge not been applied before for the
+
                                    ion channels. The addition of FAS transmembrane domain to the
+
                                    N-terminus of TRPC1 improved localization of the protein to plasma membrane in
+
                                    comparison to the unmodified TRPC1. From the confocal microscopy images
+
                                    of non-permeabilized cells we verified that the modified channel was inserted into
+
                                    plasma membrane as predicted, since in non-permeabilized cells antibodies
+
                                    stained the exposed extracellular HA-tag but not the intracellular Myc-tag (
+
                                    <ref>4</ref>
+
                                    B).
+
                                </p>
+
                                <div style="float:left; width:100%">
+
                                    <figure data-ref="4">
+
                                        <img src="https://static.igem.org/mediawiki/2016/1/13/T--Slovenia--3.2.3.png">
+
                                        <figcaption><b>Localization of fusion protein P3:FAStm:TRPC1.</b><br/>
+
                                            <p style="text-align:justify">(A) Scheme of ion channel P3:FAStm:TRPC1. (B) Ion channel P3:FAStm:TRPC1 was
+
                                            localized to plasma membrane. HEK293 cells were transfected with
+
                                            P3:FAStm:TRPC1 plasmid. 24 h after transfection cells were permeabilized
+
                                            (upper) or non-permeabilized (lower) and stained with antibodies against
+
                                            HA and Myc-tag. Localization on plasma membrane is shown with arrows.
+
</p>
+
                                        </figcaption>
+
                                    </figure>
+
                                </div>
+
 
+
                                 <p style="clear:both">In addition to the improved membrane localization, the FAS
+
                                    transmembrane domain linked to the TRPC1 presents another advantage. The TRPC1 is an
+
                                    ion
+
                                    channel with six transmembrane helices, therefore both the N- and the C-terminus of
+
                                    the protein are orientated towards the interior of the cell. By addition
+
                                    of the FAS transmembrane domain, the N-terminus of P3:FAStm:TRPC1 chimera (where P3
+
                                    stands for coiled coil, hyperlink to CC page) is exposed in the extracellular
+
                                    space and could interact with different proteins from outside the cell via the
+
                                    N-terminal tag. We reasoned that this interaction could be used to achieve a higher
+
                                    sensitivity to mechanical stimuli.</p>
+
 
+
                                <p>After we showed that the selected ion channels MscS, TRPC1 and P3:FAStm:TRPC1 are
+
                                    expressed in HEK293 and localized at the plasma membrane, we further tested their
+
                                    function as mechanosensors by exposing them to the ultrasound stimulation.
+
                                </p>
+
                            </div>
+
                            <div>
+
                                <h4><span id="us" class="section">&nbsp;</span>Ultrasound stimulation</h4><br/>
+
                                <div style="clear:both" class="ui styled fluid accordion">
+
                                    <div class="title">
+
                                        <i class="dropdown icon"></i>
+
                                        Further explanation ...
+
                                    </div>
+
                                    <div class="content">
+
                                        <p>
+
                                            Ultrasound stimulation offers potentially remarkable advantages over the
+
                                            majority of external stimuli used for targeted cell stimulation.
+
                                            Optogenetics as another
+
                                            promising approach to cell stimulation requires invasive surgery to
+
                                            implement optical fibers connected to the source of light – LED or laser
+
                                            <x-ref>Warden2014</x-ref>
+
                                            in order to target cells in tissue to activate or silence them. On the other
+
                                            hand, ultrasound offers a non-invasive approach to overcome the problems
+
                                            which appear in
+
                                            the abovementioned method. Its use has been demonstrated potentially even
+
                                            for noninvasive ultrasound therapy through an intact skull
+
                                            <x-ref>Hynynen1998</x-ref>
+
                                            .
+
                                            Previously, ultrasound had been used in several in vitro studies to directly
+
                                            stimulate clusters of neurons but also in few model organisms (among others
+
                                            <x-ref>King2013</x-ref>
+
                                            .
+
                                        </p>
+
                                    </div>
+
                                </div>
+
                                <br/>
+
                                <p>For mechanical stimulation of cells with ultrasound we designed our own unique
+
                                    experimental setup, which included the
+
                                    <a href="https://2016.igem.org/Team:Slovenia/Hardware">ultrasound device MODUSON</a>
+
                                    that we constructed connected to the unfocused transducer Olympus
+
                                    V318-SU and a 3D printed support for a transducer to fix it at a defined position
+
                                    relative to the cells. Stimulation conditions were optimized for our cell
+
                                    line and experimental setup. To measure the changes of free calcium ion
+
                                    concentration we stained cells with two fluorescent dyes Fura Red and Fluo-4.
+
                                    The combination of these two dyes enabled us to present changes in the calcium ion
+
                                    concentration as a ratio of the fluorescence intensity at two wavelengths,
+
                                    which was superior to the intensity based measurements, since it is independent of
+
                                    photobleaching and dye sequestration
+
                                </p>
+
 
+
                                <div class="ui styled fluid accordion">
+
                                    <div class="title">
+
                                        <i class="dropdown icon"></i>
+
                                        Further explanation ...
+
                                    </div>
+
                                    <div class="content">
+
                                        <p>Fura Red and Fluo-4 are visible light-excitable dyes used for ratiometric
+
                                            measurement of calcium ions which excitation maximum is at 488 nm.
+
                                            While Fluo-4 exhibits an increase in fluorescence emission at 515 nm upon
+
                                            binding of calcium ions, fluorescence emission at 655 nm of Fura Red
+
                                            decreases once the indicator binds calcium ions. By calculating the ratio of
+
                                            fluorescence emission intensities captured at 488 nm exaction
+
                                            (where the difference of fluorescence between the bound and free indicator
+
                                            is at its maximum), we could observe changes in intracellular
+
                                            calcium concentrations in real time.
+
                                        </p>
+
                                    </div>
+
                                </div>
+
                                <br/>
+
 
+
                                <p>We followed changes of calcium concentration after ultrasound stimulation in real
+
                                    time using ratiometric confocal microscopy. For processing of data we
+
                                    developed our software <a
+
                                            href="https://2016.igem.org/Team:Slovenia/Software">CaPTURE</a>, which
+
                                    automatically calculated the ratio between fluorescence
+
                                    intensities of FuraRed and Fluo-4 and presented the data as image and calculated
+
                                    values.
+
                                </p>
+
 
+
                                <p>We showed that by expressing the MscS channel, cells gained sensitivity for
+
                                    ultrasound stimulation in comparison to non-transfected cells (
+
                                    <ref>6</ref>
+
                                    ).
+
                                    Influx of calcium ions was observed at a lower rate in the case of ectopically
+
                                    expressed TRPC1 (data not shown), probably due to its poor membrane localization.
+
                                </p>
+
 
+
                                <div style="clear:both; width:50%" align="center">
+
                                    <figure data-ref="5">
+
                                        <img src=" https://static.igem.org/mediawiki/2016/f/f2/T--Slovenia--S.3.1.1.png ">
+
                                        <figcaption><b>INSERT!!!</b><br/></figcaption>
+
                                    </figure>
+
                                </div>
+
 
+
                                <div style="clear:both;" align="center">
+
                                    <figure data-ref="6">
+
                                        <img class="ui big centered image"
+
                                            src="https://static.igem.org/mediawiki/2016/2/27/T--Slovenia--3.2.5.png">
+
                                        <div style="clear:both; display:block; width: 90%; margin-right: auto; margin-left: auto">
+
                                            <div style="display: block; float: left; width: 80%;">
+
                                                <!-- experiment: 20161002 2 MscS + Gvp 200V 90s -->
+
                                                <img class="playme" id="gifGroup8"
+
                                                    src="//2016.igem.org/wiki/images/a/ad/T--Slovenia--Group71.png"
+
                                                    width="100%"
+
                                                    data-alt="//2016.igem.org/wiki/images/b/be/T--Slovenia--20160929_2_MscS_200V_110s_graf.gif">
+
                                            </div>
+
                                            <div>
+
                                                <!-- experiment: 20161002 2 MscS + Gvp 200V 90s -->
+
                                                <div style="display: block; float: left; width: 20%;">
+
                                                    <img class="gifGroup8"
+
                                                        src="//2016.igem.org/wiki/images/7/70/T--Slovenia--Group72.png"
+
                                                        width="100%"
+
                                                        data-alt="//2016.igem.org/wiki/images/8/84/T--Slovenia--20160929_2_MscS_200V_110s_Regions.gif">
+
                                                </div>
+
                                                <div style="display: block; float: left; width: 20%;">
+
                                                    <img class="gifGroup8"
+
                                                        src="//2016.igem.org/wiki/images/8/83/T--Slovenia--Group73.png"
+
                                                        width="100%"
+
                                                        data-alt="//2016.igem.org/wiki/images/8/8f/T--Slovenia--20160929_2_MscS_200V_110s_Controls.gif">
+
                                                </div>
+
                                                <div style="display: block; float: left; width: 20%;">
+
                                                    <img class="gifGroup8"
+
                                                        src="//2016.igem.org/wiki/images/0/0e/T--Slovenia--Group74.png"
+
                                                        width="100%"
+
                                                        data-alt="//2016.igem.org/wiki/images/e/e4/T--Slovenia--20160929_2_MscS_200V_110s_HeatMap.gif">
+
                                                </div>
+
                                            </div>
+
                                         </div>
+
                                        <figcaption><b> MscS channel improves sensitivity of cells for
+
                                            ultrasound.</b><br/>
+
                                            <p style="text-align:justify">(A) Schematic representation of a stimulation sequence and (B) signal
+
                                            parameters used for stimulation.
+
                                            (C) and (D )Cells expressing MscS showed increased sensitivity to ultrasound
+
                                            stimulation in comparison to the cells without exogenous mechanosensitive
+
                                            channel. HEK293 cells expressing MscS channels or control cells transfected
+
                                            with vector were stimulated with ultrasound for 10 s and calcium influx
+
                                            was recorded in real time (D) using a confocal microscope. For comparison
+
                                            cells without ectopic MscS were used. Fluo-4 (D, green line) and Fura Red
+
                                            dyes (D, red line) were used for ratiometric calcium imaging. (D) Ratio
+
                                            (blue line) was calculated from fluorescence intensities of Fura Red and
+
                                            Fluo-4
+
                                            using <a href="https://2016.igem.org/Team:Slovenia/Software">CaPTURE</a>.
+
</p>
+
                                        </figcaption>
+
                                    </figure>
+
                                </div>
+
                                <p style="clear:both">Fusion of the FAS transmembrane domain to TRPC1 did not only
+
                                    improve its membrane localization, but also significantly enhanced its sensitivity
+
                                    to ultrasound
+
                                    stimulation (
+
                                    <ref>8</ref>
+
                                    C), suggesting the importance of membrane localization in the function of
+
                                    mechanosensors.
+
                                </p>
+
 
+
                                <div style="clear:left;">
+
                                    <figure data-ref="7">
+
                                        <img onclick="resize(this);" class="ui medium image"
+
                                            src=" https://static.igem.org/mediawiki/2016/f/fd/T--Slovenia--S.3.1.2.png">
+
                                        <figcaption><b>INSERT!!!</b><br/></figcaption>
+
                                    </figure>
+
                                </div>
+
 
+
                                <div style="clear:both;" align="center">
+
                                    <figure data-ref="8">
+
                                        <img class="ui big centered image"
+
                                            src="https://static.igem.org/mediawiki/2016/8/8a/T--Slovenia--3.2.4.png">
+
                                        <div style="clear:both; display:block; width: 90%; margin-right: auto; margin-left: auto">
+
                                            <div style="display: block; float: left; width: 80%;">
+
                                                <!-- experiment: 20161002 2 MscS + Gvp 200V 90s -->
+
                                                <img class="playme" id="gifGroup9"
+
                                                    src="//2016.igem.org/wiki/images/1/10/T--Slovenia--Group51.png"
+
                                                    width="100%"
+
                                                    data-alt="//2016.igem.org/wiki/images/4/41/T--Slovenia--201607192_P3_FAS_TRPC1200V.gif">
+
                                            </div>
+
                                            <div>
+
                                                <!-- experiment: 20161002 2 MscS + Gvp 200V 90s -->
+
                                                <div style="display: block; float: left; width: 20%;">
+
                                                    <img class="gifGroup9"
+
                                                        src="//2016.igem.org/wiki/images/c/c7/T--Slovenia--Group52.png"
+
                                                        width="100%"
+
                                                        data-alt="//2016.igem.org/wiki/images/0/00/T--Slovenia--201607192_P3_FAS_TRPC1200Vregion.gif">
+
                                                </div>
+
                                                <div style="display: block; float: left; width: 20%;">
+
                                                    <img class="gifGroup9"
+
                                                        src="//2016.igem.org/wiki/images/b/be/T--Slovenia--Group53.png"
+
                                                        width="100%"
+
                                                        data-alt="//2016.igem.org/wiki/images/6/60/T--Slovenia--201607192_P3_FAS_TRPC1200Vcontrol.gif">
+
                                                </div>
+
                                                <div style="display: block; float: left; width: 20%;">
+
                                                    <img class="gifGroup9"
+
                                                        src="//2016.igem.org/wiki/images/c/cb/T--Slovenia--Group54.png"
+
                                                        width="100%"
+
                                                        data-alt="//2016.igem.org/wiki/images/b/b9/T--Slovenia--201607192_P3_FAS_TRPC1200Vheatmap.gif">
+
                                                </div>
+
                                            </div>
+
                                        </div>
+
                                        <figcaption><b>P3:FAS:TRPC1 channel improves sensitivity of cells for
+
                                            ultrasound.</b><br/>
+
                                            <p style="text-align:justify">(A) Schematic presentation of a stimulation sequence and (B) signal
+
                                            parameters used for stimulation.
+
                                            (C) and (D ) Cells expressing P3:FAS:TRPC1 showed increased sensitivity to
+
                                            ultrasound stimulation in comparison to the cells without exogenous
+
                                            mechanosensitive channel.
+
                                            HEK293 cells expressing P3:FAS:TRPC1 were stimulated with ultrasound for 10
+
                                            s and calcium influx was recorded in real time (D) using a confocal
+
                                            microscope. For comparison
+
                                            cells without ectopic MscS were used. Fluo-4 (D, green line) and Fura Red
+
                                            dyes (D, red line) were used for ratiometric calcium imaging. (D) Ratio
+
                                            (blue line) was calculated
+
                                            from fluorescence intensities of Fura Red and Fluo-4 using <a
+
                                            href="https://2016.igem.org/Team:Slovenia/Software">CaPTURE</a>.
+
</p>
+
                                        </figcaption>
+
                                    </figure>
+
                                </div>
+
                                <p style="clear:both">In order to observe mechanostimulation of cells with ectopically
+
                                    expressed mechanoreceptors we had to use high-power ultrasound, however we tested
+
                                    that the cells nevertheless
+
                                    did not lose the viability by ultrasound stimulation. Our next challenge was to
+
                                    further improve sensitivity of cells to respond to lower power ultrasound as this
+
                                    would avoid
+
                                    stimulation of any endogenous channels and limit stimulation only to the engineered.
+
 
                                 </p>
 
                                 </p>
 +
                                <p><img src="//2016.igem.org/wiki/images/0/0d/T--Slovenia--igemLogoSmall.png"
 +
                                        alt="newAtiGEM" width="30" height="30" style="display: inline;"> new at iGEM</p>
 
                             </div>
 
                             </div>
 +
                            <ul>
 +
                                <li>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) <img src="//2016.igem.org/wiki/images/d/dc/T--Slovenia--starSmall.png"
 +
                                                  alt="newAtiGEM" width="28" height="28" style="display: inline;"></li>
 +
                                <li>A custom-made ultrasound generator device was used to stimulate mammalian cells <img
 +
                                        src="//2016.igem.org/wiki/images/0/0d/T--Slovenia--igemLogoSmall.png"
 +
                                        alt="newAtiGEM" width="30" height="30" style="display: inline;"></li>
 +
                                <li>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 <img
 +
                                            src="//2016.igem.org/wiki/images/d/dc/T--Slovenia--starSmall.png"
 +
                                            alt="newAtiGEM" width="28" height="28" style="display: inline;"></li>
 +
                                <li>A cyclic proteolysis-activated luciferase reporter was experimentally verified and
 +
                                    introduced into the iGEM collection <img
 +
                                            src="//2016.igem.org/wiki/images/0/0d/T--Slovenia--igemLogoSmall.png"
 +
                                            alt="newAtiGEM" width="30" height="30" style="display: inline;"></li>
 +
                                <li>A set of four different orthogonal site-specific proteases was designed and tested
 +
                                    as split proteins with induced reconstitution in mammalian cells <img
 +
                                            src="//2016.igem.org/wiki/images/d/dc/T--Slovenia--starSmall.png"
 +
                                            alt="newAtiGEM" width="28" height="28" style="display: inline;"></li>
 +
                                <li>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 <img
 +
                                            src="//2016.igem.org/wiki/images/d/dc/T--Slovenia--starSmall.png"
 +
                                            alt="newAtiGEM" width="28" height="28" style="display: inline;"></li>
 +
                                <li>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. <img
 +
                                            src="//2016.igem.org/wiki/images/d/dc/T--Slovenia--starSmall.png"
 +
                                            alt="newAtiGEM" width="28" height="28" style="display: inline;"></li>
 +
                            </ul>
 +
                        </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>
 
                         </div>
 
                     </div>
 
                     </div>
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Latest revision as of 13:50, 19 October 2016

Home

  Sonicell

For the therapy of diseases such as diabetes, we require a system of drug delivery that is fast, controllable and noninvasive. Engineering cells to produce drugs in the tissue is a promising direction. However, in most genetic circuits designed by synthetic biology the response is delayed due to the need to transcripe and translate the mediators. 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

 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.