Difference between revisions of "Team:Peking/Design"

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     <head>
 
     <head>
 
         <!--- Basic Page Needs========================================================================= -->
 
         <!--- Basic Page Needs========================================================================= -->
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         <meta charset="utf-8">
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         <meta charset="utf-8"/>
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            <title>Overview</title>
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        <title></title>
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            <meta name="viewport" content="width=device-width, initial-scale=1.0, maximum-scale=1.2, user-scalable=yes" />
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        <meta name="viewport" content="width=device-width, initial-scale=1.0, maximum-scale=1.2, user-scalable=yes" />
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            <meta name="description" content="Wiki of Peking iGEM 2016" />
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        <meta name="description" content="Wiki of Peking iGEM 2016" />
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            <meta name="author" content="Li Jiamian & Wang Yuqing">
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        <meta name="author" content="Li Jiamian & Wang Yuqing"/>
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                <meta http-equiv="Content-Type" content="text/html; charset=utf-8" />
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        <meta http-equiv="Content-Type" content="text/html; charset=utf-8" />
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                <!-- Mobile Specific Metas===================================================================== -->
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        <!-- Mobile Specific Metas===================================================================== -->
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                <meta name="viewport" content="width=device-width, initial-scale=1, maximum-scale=1">
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        <meta name="viewport" content="width=device-width, initial-scale=1, maximum-scale=1"/>
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                    <!-- Fix  Overwrite the original iGEM style=================================================== -->
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        <!-- Fix  Overwrite the original iGEM style=================================================== -->
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                    <link href="https://2016.igem.org/Template:Peking/css/fix?action=raw&ctype=text/css" rel="stylesheet" />
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        <link href="https://2016.igem.org/Template:Peking/css/fix?action=raw&ctype=text/css" rel="stylesheet" />
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                    <!-- CSS======================================================================================= -->
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        <!-- CSS======================================================================================= -->
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                    <link href="https://2016.igem.org/Template:Peking/css/bootstrap_min?action=raw&ctype=text/css" rel="stylesheet" />
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        <link href="https://2016.igem.org/Template:Peking/css/bootstrap_min?action=raw&ctype=text/css" rel="stylesheet" />
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                    <link href="https://2016.igem.org/Template:Peking/css/style?action=raw&ctype=text/css" rel="stylesheet" />
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        <link href="https://2016.igem.org/Template:Peking/css/style?action=raw&ctype=text/css" rel="stylesheet" />
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                    <!-- CSS======================================================================================= -->
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        <!-- CSS======================================================================================= -->
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                    <link rel="stylesheet" href="https://2016.igem.org/Template:Peking/css/default?action=raw&ctype=text/css">
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        <link rel="stylesheet" href="https://2016.igem.org/Template:Peking/css/default?action=raw&ctype=text/css"/>
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                        <link rel="stylesheet" href="https://2016.igem.org/Template:Peking/css/layout?action=raw&ctype=text/css">
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        <link rel="stylesheet" href="https://2016.igem.org/Template:Peking/css/layout?action=raw&ctype=text/css"/>
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                            <link rel="stylesheet" href="https://2016.igem.org/Template:Peking/css/media-queries?action=raw&ctype=text/css">
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        <link rel="stylesheet" href="https://2016.igem.org/Template:Peking/css/media-queries?action=raw&ctype=text/css"/>
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                                <link rel="stylesheet" href="https://2016.igem.org/Template:Peking/css/priorstyle?action=raw&ctype=text/css">
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        <link rel="stylesheet" href="https://2016.igem.org/Template:Peking/css/notebook_panel?action=raw&ctype=text/css"/>
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                                    <style>
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        <style>
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                                        .home_img{
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            .texttitle{
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                                            padding: 10px;
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                color: #11abb0;
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                                            margin: 10px;
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                font-size: 38px;
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                                            width: 420px;
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                line-height: 48px;
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                                            float: left;
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                margin-bottom: 12px;
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                                            height: 300px;
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                font-family: raleway-bold, sans-serif !important;
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                                        }
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                background: transparent;
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                letter-spacing: 3px;
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                                        padding-top: 20px;
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                text-transform: uppercase;
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                                        margin: 10px;
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                font-weight: 350;
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                                        float: left;
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                text-align: center;
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                                        height: 300px;
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                padding-top:40px;
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        sup{font-size:11px;}
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                                        margin-bottom: 12px;
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        }
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        figure{margin-top:40px;margin-bottom:40px;height:auto;}
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                                    .references{margin-top:150px;margin-bottom:40px;}
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                                    .references p{font-size:14px !important; color:#666161 !important;}
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                                    .classic-title {font-weight: 300;}
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                                    .classic-title span {
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                                        </style>
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                                    </head>
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     <body>
 
     <body>
 +
        <!--sidebar 引用==============================================================================-->
 +
        <style>
 +
            #primary span{
 +
                display:block;
 +
                word-break:break-all
 +
            }
 +
       
 +
        #page-wrap {
 +
            width: 25%;
 +
            margin: 0px;
 +
            position: relative;
 +
        }
 +
       
 +
        #sidebar {
 +
            width: 25%;
 +
            margin-left: 0px;
 +
        }
 +
        @media (min-width:1024px){
 +
            #sidebar{position:relative;top:120px;max-width:200px;}}
 +
        @media (max-width: 1023px){
 +
            #sidebar{display:none;
 +
            }
 +
            #page-wrap{display:none;}
 +
        }
 +
        </style>
 +
       
 +
       
 +
        <script type="text/javascript">
 +
            function menuFixed(id){
 +
                var obj = document.getElementById(id);
 +
                var _getHeight = obj.offsetTop;
 +
               
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                window.onscroll = function(){
 +
                    changePos(id,_getHeight);
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                }
 +
            }
 +
        function changePos(id,height){
 +
            var obj = document.getElementById(id);var windowBottom = $(window).scrollTop() + $(window).innerHeight();
 +
            if(w>=1024){
 +
                if($(window).scrollTop() + $(window).height() > $(document).height() - 230){
 +
                    $('#sidebar').fadeOut("fast");}else{$('#sidebar').fadeIn("fast");}
 +
            }
 +
            var scrollTop = document.documentElement.scrollTop || document.body.scrollTop -230;
 +
            var windowBottom = $(window).scrollTop() + $(window).innerHeight();
 +
            var w = window.innerWidth;
 +
           
 +
            if(scrollTop < height){ obj.style.position = 'relative';
 +
            }else{
 +
                obj.style.position = 'fixed';
 +
            }
 +
        }
 +
        </script>
 +
       
 +
        <script type="text/javascript">
 +
            window.onload = function(){
 +
                menuFixed('sidebar');
 +
            }
 +
        </script>
 +
        <script>
 +
            function naver(id){
 +
                var obj=document.getElementById(id);
 +
                var oPos=obj.offsetTop;
 +
                return window.scrollTo(0,oPos+250);
 +
            }
 +
        </script>
 +
        <!--sidebar 引用 end ==============================================================================-->
 +
        <!--panel 引用==================================================================================-->
 +
        <style type="text/css">
 +
            .panel-default .panel-heading a{
 +
                text-decoration: none;
 +
                display:block;
 +
                padding:10px;
 +
            }
 +
        .panel-heading.panel-title{
 +
            text-decoration: none;
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            padding-top:0px;
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            padding-bottom:0px;
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            padding-left:0px;
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            padding-right:0px;
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            text-align:center;
 +
            font-size:19px;
 +
           
 +
        }
 +
        a[aria-expanded="true"] {
 +
            background-color:rgba(70, 73, 76, 0.95);
 +
            text-decoration: none;
 +
            color:white;
 +
        }
 +
       
 +
        .panel-default .panel-heading a[aria-expanded="false"]{
 +
            -o-transition: background-color 1s linear;
 +
            -moz-transition: background-color 1s linear;
 +
            -khtml-transition: background-color 1s linear;
 +
            -webkit-transition: background-color 1s linear;
 +
            -ms-transition: background-color 1s linear;
 +
            transition: background-color 1s linear;
 +
        }
 +
        .panel-default .panel-heading a[aria-expanded="false"]:hover{
 +
            background-color:rgba(70, 73, 76, 0.95);
 +
            text-decoration: none;
 +
            color:white;
 +
        }
 +
        .panel-default .panel-heading a[aria-expanded="true"]{
 +
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 +
            transition: opacity 0.7s linear;
 +
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 +
        .panel-default .panel-heading a[aria-expanded="true"]:hover{
 +
            opacity:0.7;
 +
        }
 +
       
 +
        .coll p a{
 +
            color:#5c9085 !important;
 +
        }
 +
        .coll p a:hover{
 +
            color:#11abb0 !important;
 +
        }
 +
        .coll {
 +
            width:100%;
 +
            float:left;
 +
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 +
        .coll .info {
 +
            width:6%;
 +
            font-size:11px;
 +
            color:#a4a4a4;
 +
            margin-top:0px;
 +
            float:left;
 +
           
 +
        }
 +
        .coll .content {
 +
            width:94%;
 +
            float:left;
 +
            margin-top:-3px;
 +
        }
 +
        .coll .ordi {
 +
            width;100%;
 +
            margin:0px 0px 0px 0px;
 +
            font-size:35px;
 +
            font-style:italic;
 +
            float:left;
 +
            color:#11abb0;
 +
            opacity:0.8;
 +
        }
 +
       
 +
            </style>
 +
       
 +
        <script type="text/javascript">
 +
            $(document).ready(function(){
 +
                              $("#button1").click(function(){
 +
                                                  $(".panel-collapse").collapse("show");
 +
                                                  });
 +
                              });
 +
                              $(document).ready(function(){
 +
                                                $("#button2").click(function(){
 +
                                                                    $(".panel-collapse").collapse("hide");
 +
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 +
                                                });
 +
                                                $("#notebook").addClass("navbar-active");
 +
            </script>
 +
        <!--panel 引用 end ==================-->
 +
       
 
           <!-- Navigation -->
 
           <!-- Navigation -->
 
         <div id="navigation" class="navbar navbar-fixed-top">
 
         <div id="navigation" class="navbar navbar-fixed-top">
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         </div>
 
         </div>
 
         <!--/Navigation -->
 
         <!--/Navigation -->
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−
 
 
          
 
          
 +
        <!-- Page Title======================================================================== -->
 +
        <div id="page-title">
 +
            <div class="row">
 +
                <div class="twelve columns centered text-center">
 +
                    <h1>Design</h1>
 +
                    <p class="title1" style="text-align:center">While people are constantly exploring the world, the greatest pursuit is to remould the world. While the ‘phase separation’ in cells is under investigation and in a research boom, the scientific community hopes the phenomenon ‘worth of millions of dollars’ to be artificially designed to enhance original functions and even acquire new functions. Our team, Peking iGEM 2018 go all out to overcome the challenge: fulfill phase separation in cells and synthesize membraneless organelles.</p>
 +
                </div>
 +
            </div>
 +
        </div><!-- Page Title End-->
 
          
 
          
−
         <div class="content-outer">
+
       
−
             <div id="page-content" class="row page">
+
         <div id="page-content" class="row page">
 +
             <div id="primary" class="twelve columns">
 +
                <section>
 +
                    <div class="row">
 +
                       
 +
                       
 +
                       
 +
                       
 +
                        <div class="three columns">
 +
                            <div id="page-wrap">
 +
                                <div id="sidebar" style="color:#000000">
 +
                                    <h4><a href="javascript:void(0);" onclick="naver('A')">Overview</a></h4>
 +
                                    <h4><a href="javascript:void(0);" onclick="naver('B')">Phase&nbsp;Separation&nbsp;System</a></h4>
 +
                                    <h4><a href="javascript:void(0);" onclick="naver('C')">Functional&nbsp;Organelles</a></h4>
 +
                                    <ul>
 +
                                        <li><a href="javascript:void(0);" onclick="naver('C1')">Spontaneous</a></li>
 +
                                        <li><a href="javascript:void(0);" onclick="naver('C2')">The&nbsp;formation</a></li>
 +
                                        <li><a href="javascript:void(0);" onclick="naver('C3')">Spontaneous</a></li>
 +
                                        <li><a href="javascript:void(0);" onclick="naver('C4')">The&nbsp;formation</a></li>
 +
                                    </ul>
 +
                                    <h4><a href="javascript:void(0);" onclick="naver('D')">Perspective</a></h4>
 +
                                </div>
 +
                            </div>
 +
                        </div>
 +
                       
 +
                       
 +
                       
 +
                       
 +
                        <div class="nine columns">
 +
                           
 +
                            <div class="texttitle">Overview
 +
<a id="A"></a></div>
 +
                            <hr style="border:2px dashed; height:2px" color="#666666">
  
 +
                            <div class="coll">
 +
                               
 +
                                <div class="content">
 +
                                    <p>The aim of our project is to build a synthetic organelle based on phase separation as a multifunctional platform. Based on the principle of multivalence and interaction, we fused interactional modules into homo-oligomeric tags (HOtags) to form granules in S. cerevisiae.</p>
 +
                                </div>
 +
                            </div>
 +
                            <div class="coll">
 +
                               
 +
                                <div class="content">
 +
                                    <p>We have built spontaneous and induced synthetic organelles by specific interaction modules, so that we can control the formation process by different ways for demands in biological engineering. Then we characterized the kinetics and properties of synthetic organelles theoretically and experimentally. These results confirm the potential of synthetic organelles in synthetic biology.</p>
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                                <div class="content">
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                                    <p>It inspired us to propose some specific applications of our synthetic organelles, including organization hub, sensor, and metabolism regulator. We have verified the feasibility of them by loading GFP-nanobody module, NAD+ sensor module and carotene production module to the whole system.</p>
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                                    <p>We believe that our work has reached the medal requirements of demonstration as we have confirmed that our synthetic organelles can be formed in vivo and deliver a range of functions both for engineering and research due to their amazing properties. The concrete demonstration of the whole platform is shown below. You can see more details of experiments and modeling in our <a href="https://2018.igem.org/Team:Peking/Results"/>Data Page</a> and <a href="https://2018.igem.org/Team:Peking/Model"/>Modeling</a></p><br/><br/><br/>     
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<div class="texttitle">Phase Separation System
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                                    <div class="ordi">1.</div>
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                                <div class="content">
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                                    <h3>Spontaneous and induced synthetic organelles can be formed by phase separation</h3>
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Design
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                                <div class="content">
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By SSY
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                                    <p>Our basic system consists of two components of synthetic organelles. Either of them has a specific HOtag to form homo-oligomers. We expect that they are able to form synthetic organelles due to the principles of phase separation. To verify the feasibility of the design, we fused two fluorescence proteins with the two components of synthetic organelles (Figure1.a) so that we can observe the self-organization of components and the formation of granules under fluorescence microscope.</p>
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While people are constantly exploring the world, the greatest pursuit is to remould the world. While the ‘phase separation’ in cells is under investigation and in a research boom, the scientific community hopes the phenomenon ‘worth of millions of dollars’ to be artificially designed to enhance original functions and even acquire new functions. Our team, Peking iGEM 2018 go all out to overcome the challenge: fulfill phase separation in cells and synthesize membraneless organelles.
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                                    <p>We used SUMO-SIM interaction module to build a spontaneous organelle. When two components are expressed in yeasts, granules with the two fluorescence proteins can be observed in vivo (Figure1.b). </p>
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一、 Overall design
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Then we put forward two questions: Why phase separation in cells can produce membraneless organelles? And how can we design our system to fulfill its intended functions?
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                                    <p>Meanwhile, by rapamycin induced interaction module, FKBP-Frb, we have built an inducible organelle. We can see granules occurs in yeasts within minutes after adding the inducer.</a> </p>
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Like oil in water, the contents of cells can separate into droplets. According to physical principles, the process where material self-assemble into organelles is described as ‘phase separation’, which is the conversion of a single-phase system into a multiphase system. In general, materials flow to regions with low chemical potential instead of low concentration. Finally, the components no longer distribute uniformly but form granules locally which are organelles in the cell.  
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That is to say, the main work to synthesize an organelle is to fulfill phase separation in a cell. Then, how can we do it? Composition can switch rapidly through multivalency. And our design was inspired by recent works showing that multivalency drives protein phase separation and formation of synthetic organelles. What’s more, we take our inspiration from existing life systems and previous works. For example, Intrinsic Disordered Regions are the symbol of massive phase separation in the cell. They interact with each other through van der Waals force, hydrophobic effect and electrostatic attraction. And there are many interactions like this in nature, such as FKBP and FRB, SUMO and SIM, SH3 and PRM, phyB and PIF6. Thus, we can make good use of them to induce our designed organelles and regulate them variously.
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Figure1.a The basic design of synthetic organelles with florescence reporters. <img src="https://static.igem.org/mediawiki/2018/8/80/T--Peking--toolbar_logo.jpeg" style="width:100%;" alt="">（这里可能需要一张cartoon的设计图）
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In conclusion，multivalency drives protein’s self-assembly and interaction binds the parts together. It means, interaction can induce phase separation and multivalency can make larger assemblies, which are two essential elements in our design and ensure the formation of synthetic organelles.
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            b, c fluorescence images of spontaneous organelles (SUMO-SIM based) and inducible synthetic organelles (FKBP-Frb based, after adding 10000 nM rapamycin)<br/><br/>
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Figure 1  Overall design
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二、 Multivalency
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To design multivalent modules, it is not ideal to use multiple repeat domains, which will not only make the protein extremely large and bring difficulty to molecular cloning, but also may be problematic for making transgenic animals. Thus, instead of using multiple repeats, we turned to de novo-designed homo-oligomeric coiled coils. And we named these coiled coils as HOTag (Homo-Oligomeric Tag). They are short peptides, ~30 amino acids, therefore they are ideal tags to introduce multivalency. There are seven coiled coils previously characterized in protein de novo design studies. It has been proved by previous work of Shu Xiaokun’s lab that HOTag3 and HOTag6 are most robust in driving protein droplet formation over a wide range of protein concentrations, so we chose them.
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Figure 2  Coiled-coil assemblies and helical bundles.
 
  
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三、 Interaction
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                            <div class="coll">
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To design interaction modules, we tried a lot of components and we fused them to the N-terminus of HOTag3 or HOTag6. Some of them are spontaneous and some are inducible. And we can regulate them through various kinds of inducers and different intensities of promoters. 
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                                    <div class="ordi">2.</div>
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                                <div class="content">
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                                    <h3>The formation of organelles has flexible but predictable properties and kinetics in different conditions</h3>
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                            <div class="coll">
  
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(一) SUMO and SIM
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                                <div class="content">
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Post-translational modifications by the small ubiquitin-like modifier (SUMO) are crucial events in cellular response to radiation and a wide range of DNA-damaging agents. Previous studies have shown that SUMO mediates protein-protein interactions by binding to a SUMO-interacting motif (SIM) on receptor proteins. And recent studies have shown that a protein with ten repeats of human SUMO3 (polySUMO) and a protein with ten repeats of SIM (polySIM) can phase separate in vitro. Therefore, we chose SUMO3 and SIM as a pair of interaction modules and they can drive the formation of synthetic organelles spontaneously. For plasmid construction, in order to make synthetic organelles visible, we chose mCherry (red fluorescent protein) and yEGFP (yeast-enhanced green fluorescent protein) as reporters. Then, we fused mCherry between the C-terminus of SIM and the N-terminus of HOTag6. Similarly, we fused yEGFP between the C-terminus of SUMO and the N-terminus of HOTag3. We transformed them into yeast and proved that they can stably express. If it work, we will find red granules colocalize with green granules in cells under fluorescence microscope.  
+
                                    <p>Then we combined <a href="https://2018.igem.org/Team:Peking/Phase_Separation_M"/>modeling of phase separation</a> and experiment to research the kinetics of the organelles formation process expecting that a well-characterized system can reach its whole potential in complex applications. </p>
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There is a pattern diagram composing SUMO and SIM.
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Figure 3  Interaction of SUMO and SIM
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(二) FKBP and Frb
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                                <div class="content">
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The interaction between FKBP and FRB can be robustly induced by rapamycin. Rapamycin is a 31-membered macrolide antifungal antibiotic. It binds with high affinity (Kd=0.2nM) to the 12-kDa FK506 binding protein (FKBP), as well as to a 100-amino acid domain of the mammalian target of rapamycin (mTOR) known as FKBP-rapamycin binding domain (Frb). Thus, we chose them as a pair of interaction modules. And we assembled them on to yeast plasmid as the same as the construction of SUMO and SIM and transformed them into yeast. if we add rapamycin to the yeast, we will see red granules colocalize with green granules in cells under fluorescence microscope. Synthetic organelles come true!
+
                                    <p>As the model predicts, the concentration of components and the interaction strength affect the kinetics of phase separation. First we controlled the expression levels of components by using several stable or inducible promoters and observe the system's behavior. We found that the formation of organelles happened in specific promoter combinations and can be controlled by inducible promoters. The analysis result does not only fit well with the simulation, but provides potential methods to control the organelles in applications. </p>
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                            <div class="coll">
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<br/>
 +
Figure2 (a) Phase diagram of a phase separation system with three components(simulation). To fit our system, the x-axis and the y-axis stands for the two components in the granules. The asymmetry comes from the assumption that the two components have different interactions with water.
 +
(b) Fluorescence movies of different promoter combinations of FKBP-Frb mediated system after adding rapamycin. Only in specific combinations, synthetic organelles can be formed by phase separation.
 +
(c) The formation process of SUMO-SIM mediated synthetic organelles can be controlled by inducible promoters. While the expression of Tet07-SIM-mCherry-HoTag6 is induced by dox gradually, the granules will occur abruptly in some time.<br/><br/>
  
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Figure 4  Interaction of FKBP and FRB induced by rapamycin
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                                <div class="content">
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                                    <p>The strength of interaction modules can be also controlled. In the rapamycin-induced organelle system, changing the concentration of rapamycin will affect the apparent value of K, a parameter reflecting the interaction strength in our model. In a gradient rapamycin-inducing experiment, the delay time from adding inducer to granules formation was found to be shorter when concentration of rapamycin increases. So we have confirmed the influence of two parameters in models and increased the flexibility of our synthetic organelles.</p>
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                            <div class="coll">
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<br/>
 +
Figure3 (a) A simulation of organelle formation process in different interaction strength of components.
 +
(b) The speed of FKBP-Frb mediated organelle formation increases with the increasing concentration of rapamycin.
 +
<br/><br/>
  
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(三) Optogenetic control
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All the interaction modules described above are irreversible.
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                            <div class="coll">
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Now, this problem can be solved be the Arabidopsis red light-inducible phytochrome (PHYB-PIF) system, which comprises the phytochrome B (PHYB) protein and phytochrome interaction factor (PIF; PIF3 or PIF6). These two domains are induced to bind under far infrared light and the binding is reversed within seconds of exposure to infrared light but is otherwise stable for hours in the dark. What’s more, the phytochrome system has a 10-100  larger dynamic range than the cryptochrome and LOV-based systems, and the affinity of its light-gated interaction is tighter than others. Therefore, we chose PHYB and PIF6 as a pair of interaction modules. Then, we assembled them onto yeast plasmid in the same way as the construction of SUMO and SIM and transformed them into yeast. We suppose SPOT can form and disappear under the regulation of far infrared light.   
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                                <div class="content">
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Figure 7 Interaction of PhyB and PIF
+
                                    <p>We also tried to characterized other properties, like the liquid-like property of the synthetic organelles, as they may affect the functions. See more details about our characterizations in <a href="https://2018.igem.org/Team:Peking/Phase_Separation_D"/>DataPage Phase separation</a>.</p><br/><br/><br/>
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 +
                            </div>
  
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(四) Two function sites
+
                       
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Now, we have designed artificial membraneless organelles. But how can we fulfill intended functions with them? Here, we propose two ideas. We reserve two sites for implement functions, where function modules like enzymes in metabolism, proteins in signaling pathway and transcription factors in transcription can be loaded.
+
                           
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Direct integration into the skeleton
+
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Just as we characterize synthetic organelles with fluorescent proteins, we can fuse function modules between the C-terminus of interaction modules and the N-terminus of HOTags. Then, the function modules can be fuse into S to fulfill intended functions.
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Figure 8  Integrate function modules to the skeleton
+
  
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Targeting GFP with nanobody
+
                            <div class="texttitle">Functional Organelles
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We introduce a magic protein, anti-GFP nanobody, which is very small (only 13-kDa, 1.5nm 2.5nm) and a high-affinity (0.59nM) camelid antibody to GFP. So we can use its characteristic to improve our designs. We can fuse GFP between the C-terminus of interaction modules and the N-terminus of HOTags, and fuse function modules to the C-terminus of anti-GFP nanobodies. Then, with the help of interaction between anti-GFP nanobodies and GFP, SPOT can load function modules, expected functions can be realized. You may ask: How does anti-GFP nanobody improve the design? Firstly, it will not make the protein extremely large and will reduce the effect on the structure of function modules, which can ensure the quality of functions. Secondly, it can bring components not belonging to the original structure to synthetic organelles, which can extend the function of synthetic organelles. Thirdly, it is easy to regulate the expression of target proteins. So you can see, nanobodies may do better and give you a surprise!
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Figure 9  Interaction of anti-GFP nanobody and GFP
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                                    <p>Since SPOT can form in the cell and be controlled, we go further to consider the functions of SPOT. The functions of SPOT can be descripted in three catalogs: Spatial segmentation, Sensor and metabolic regulation. We verified the spatial segmentation with the condensation of substrates, also we can load the protein we want by fusing it with nanobody. We then verified the sensor with detecting rapamycin and ABA, which shows strong relativity between the concentration and the proportion of yeasts with SPOT. To find the law behind metabolism in the SPOT, we fuse the enzymes that can produce β-carotene into SPOT and measure the difference between with or without SPOT in produce of β-carotene.</p>
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                            <div class="coll">
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Figure4 (organization hub)
 +
Design of GFP-nanobody based system
 +
fluorescence images of GFP-nanobody based system
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Figure5 (sensor)
 +
(a)~(?) fluorescence images of sensor based system
 +
Figure6 (metabolism)
 +
Characterization of carotene production system
 +
（phase内和phase外的胡萝卜素生产实验）<br/><br/><br/><br/><br/>
  
−
四、 Conclusion
+
                            </div>
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We fulfilled phase separation in vivo and synthesized membraneless organelles. And the main work to synthesize an organelle is to fulfill phase separation in a cell, so we stress the importance of interactions and multivalency. For these two aspects, we gave our ideas and the feasibility was analyzed. At last, we proposed two ideas to implement functions. We believe that in the near future, “millions of dollars” will no longer be a dream!
+
                           
  
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References
+
                            <div class="texttitle">Perspective
−
1. Yin P, Fan H, Hao Q, et al. Structural insights into the mechanism of abscisic acid signaling by PYL proteins[J]. Nature Structural & Molecular Biology, 2009, 16(12):1230-1236.
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2. Park S Y, Fung P, Nishimura N, et al. Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins[J]. Science, 2009, 324(5930):1068-1071.
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3. 冯婵莹, 王永飞. 植物脱落酸PYR/PYL/RCAR受体[J]. 生命的化学, 2015(6):721-726.
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4. Nambara E, Marion-Poll A. Abscisic acid biosynthesis and catabolism.[J]. Annual Review of Plant Biology, 2005, 56(56):165-185.
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5. Hirano K, Ueguchi-Tanaka M, Matsuoka M. GID1-mediated gibberellin signaling in plants[J]. Trends in Plant Science, 2008, 13(4):192-199.
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                                    <p>SPOT has been well verified and has various functions. And in the future, this modular system will have great potential in science and practice using. SPOT can change the modules to gain more different properties like diverse inducing method, we can also use it as a platform and then load other protein with some interactions like the interaction between nanobody and GFP. What’s more, we might have the ability to form differernt SPOTs in the cell and regulate them respectively. The functions of SPOT can also diverse. We can build a real time sensor for molecule in living cells to monitoring the concentration changing in environment or in cells. More metabolism pathway can be test in SPOT and we will find some laws of the function of regulate the metabolism. To be summary, more achievement is coming true with SPOT.</p>
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6. Nelis S, Conti L, Zhang C, et al. A functional Small Ubiquitin-like Modifier (SUMO) interacting motif (SIM) in the gibberellin hormone receptor GID1 is conserved in cereal crops and disrupting this motif does not abolish hormone dependency of the DELLA-GID1 interaction[J]. Plant Signaling & Behavior, 2015, 10(2):e987528.
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                                </div>
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7. Ueguchi-Tanaka M, Nakajima M, Motoyuki A, et al. Gibberellin receptor and its role in gibberellin signaling in plants.[J]. Annual Review of Plant Biology, 2007, 58(1):183-198.
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8. Ries J, Kaplan C, Platonova E, et al. A simple, versatile method for GFP-based super-resolution microscopy via nanobodies.[J]. Nature Methods, 2012, 9(6):582-584.
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9. Caussinus E, Kanca O, Affolter M. Fluorescent fusion protein knockout mediated by anti-GFP nanobody[J]. Nature Structural & Molecular Biology, 2011, 19(1):117-121.
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10. Zhang K, Cui B. Optogenetic control of intracellular signaling pathways[J]. Trends in Biotechnology, 2015, 33(2):92-100.
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11. Buckley C, Moore R, Reade A, et al. Reversible Optogenetic Control of Subcellular Protein Localization in a Live Vertebrate Embryo[J]. Developmental Cell, 2016, 36(1):117.
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12. Banaszynski L A, And C W L, Wandless T J. Characterization of the FKBP·Rapamycin·FRB Ternary Complex  [J. Am. Chem. Soc. 2005, 127, 4715−4721].[J]. Journal of the American Chemical Society, 2006, 128(49).
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13. Woolfson D N, Bartlett G J, Burton A J, et al. De novo protein design: how do we expand into the universe of possible protein structures?[J]. Current Opinion in Structural Biology, 2015, 33:16-26.
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14. Banani S F, Rice A M, Peeples W B, et al. Compositional Control of Phase-Separated Cellular Bodies[J]. Cell, 2016, 166(3):651-663.
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15. Zhang Q, Huang H, Lu Q, et al. Visualizing Dynamics of Cell Signaling InVivo, with a Phase Separation-Based Kinase Reporter[J]. Molecular Cell, 2018, 69(2):347.
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Revision as of 11:31, 14 October 2018

Design

While people are constantly exploring the world, the greatest pursuit is to remould the world. While the ‘phase separation’ in cells is under investigation and in a research boom, the scientific community hopes the phenomenon ‘worth of millions of dollars’ to be artificially designed to enhance original functions and even acquire new functions. Our team, Peking iGEM 2018 go all out to overcome the challenge: fulfill phase separation in cells and synthesize membraneless organelles.

Overview

The aim of our project is to build a synthetic organelle based on phase separation as a multifunctional platform. Based on the principle of multivalence and interaction, we fused interactional modules into homo-oligomeric tags (HOtags) to form granules in S. cerevisiae.

We have built spontaneous and induced synthetic organelles by specific interaction modules, so that we can control the formation process by different ways for demands in biological engineering. Then we characterized the kinetics and properties of synthetic organelles theoretically and experimentally. These results confirm the potential of synthetic organelles in synthetic biology.

It inspired us to propose some specific applications of our synthetic organelles, including organization hub, sensor, and metabolism regulator. We have verified the feasibility of them by loading GFP-nanobody module, NAD+ sensor module and carotene production module to the whole system.

We believe that our work has reached the medal requirements of demonstration as we have confirmed that our synthetic organelles can be formed in vivo and deliver a range of functions both for engineering and research due to their amazing properties. The concrete demonstration of the whole platform is shown below. You can see more details of experiments and modeling in our Data Page and Modeling




Phase Separation System
1.

Spontaneous and induced synthetic organelles can be formed by phase separation

Our basic system consists of two components of synthetic organelles. Either of them has a specific HOtag to form homo-oligomers. We expect that they are able to form synthetic organelles due to the principles of phase separation. To verify the feasibility of the design, we fused two fluorescence proteins with the two components of synthetic organelles (Figure1.a) so that we can observe the self-organization of components and the formation of granules under fluorescence microscope.

We used SUMO-SIM interaction module to build a spontaneous organelle. When two components are expressed in yeasts, granules with the two fluorescence proteins can be observed in vivo (Figure1.b).

Meanwhile, by rapamycin induced interaction module, FKBP-Frb, we have built an inducible organelle. We can see granules occurs in yeasts within minutes after adding the inducer.

Figure1.a The basic design of synthetic organelles with florescence reporters. （这里可能需要一张cartoon的设计图） b, c fluorescence images of spontaneous organelles (SUMO-SIM based) and inducible synthetic organelles (FKBP-Frb based, after adding 10000 nM rapamycin)

2.

The formation of organelles has flexible but predictable properties and kinetics in different conditions

Then we combined modeling of phase separation and experiment to research the kinetics of the organelles formation process expecting that a well-characterized system can reach its whole potential in complex applications.

As the model predicts, the concentration of components and the interaction strength affect the kinetics of phase separation. First we controlled the expression levels of components by using several stable or inducible promoters and observe the system's behavior. We found that the formation of organelles happened in specific promoter combinations and can be controlled by inducible promoters. The analysis result does not only fit well with the simulation, but provides potential methods to control the organelles in applications.


Figure2 (a) Phase diagram of a phase separation system with three components(simulation). To fit our system, the x-axis and the y-axis stands for the two components in the granules. The asymmetry comes from the assumption that the two components have different interactions with water. (b) Fluorescence movies of different promoter combinations of FKBP-Frb mediated system after adding rapamycin. Only in specific combinations, synthetic organelles can be formed by phase separation. (c) The formation process of SUMO-SIM mediated synthetic organelles can be controlled by inducible promoters. While the expression of Tet07-SIM-mCherry-HoTag6 is induced by dox gradually, the granules will occur abruptly in some time.

The strength of interaction modules can be also controlled. In the rapamycin-induced organelle system, changing the concentration of rapamycin will affect the apparent value of K, a parameter reflecting the interaction strength in our model. In a gradient rapamycin-inducing experiment, the delay time from adding inducer to granules formation was found to be shorter when concentration of rapamycin increases. So we have confirmed the influence of two parameters in models and increased the flexibility of our synthetic organelles.


Figure3 (a) A simulation of organelle formation process in different interaction strength of components. (b) The speed of FKBP-Frb mediated organelle formation increases with the increasing concentration of rapamycin.

We also tried to characterized other properties, like the liquid-like property of the synthetic organelles, as they may affect the functions. See more details about our characterizations in DataPage Phase separation.




Functional Organelles

Since SPOT can form in the cell and be controlled, we go further to consider the functions of SPOT. The functions of SPOT can be descripted in three catalogs: Spatial segmentation, Sensor and metabolic regulation. We verified the spatial segmentation with the condensation of substrates, also we can load the protein we want by fusing it with nanobody. We then verified the sensor with detecting rapamycin and ABA, which shows strong relativity between the concentration and the proportion of yeasts with SPOT. To find the law behind metabolism in the SPOT, we fuse the enzymes that can produce β-carotene into SPOT and measure the difference between with or without SPOT in produce of β-carotene.

Figure4 (organization hub) Design of GFP-nanobody based system fluorescence images of GFP-nanobody based system Figure5 (sensor) (a)~(?) fluorescence images of sensor based system Figure6 (metabolism) Characterization of carotene production system （phase内和phase外的胡萝卜素生产实验）




Perspective

SPOT has been well verified and has various functions. And in the future, this modular system will have great potential in science and practice using. SPOT can change the modules to gain more different properties like diverse inducing method, we can also use it as a platform and then load other protein with some interactions like the interaction between nanobody and GFP. What’s more, we might have the ability to form differernt SPOTs in the cell and regulate them respectively. The functions of SPOT can also diverse. We can build a real time sensor for molecule in living cells to monitoring the concentration changing in environment or in cells. More metabolism pathway can be test in SPOT and we will find some laws of the function of regulate the metabolism. To be summary, more achievement is coming true with SPOT.