Difference between revisions of "Team:USTC"

 
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{{USTC}}
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<html>
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<div class="loading">
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<div class="row" style="margin-bottom:46px;">
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                    <a href="#Overview" class="white-text active waves-effect waves-light">Overview</a>
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      </div>
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                    <a href="#ROSE" class="white-text waves-effect waves-light">ROSE</a>
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      </div>
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    </div>
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                    <a href="#CACCI" class="white-text waves-effect waves-light">CACCI</a>
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</div>
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<div class="row">
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<div class="right">
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     <div class="col offset-m1 offset-l2 s12 m10 l8" >
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  <ul id="menu" hidden>
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        <div id="Overview" class="row">
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     <li data-menuanchor="welcomePage" class="active"><a href="#welcomePage">Welcome</a></li>
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            <div class="card hoverable ">
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    <li data-menuanchor="introPage"><a href="#introPage">Brief Intro</a></li>
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                <div class="row">
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    <li data-menuanchor="constitutionPage"><a href="#constitutionPage">Constitutions</a></li>
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                    <div class="col s12 m9">
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    <li data-menuanchor="tutorialPage"><a href="#tutorialPage">Tutorials</a></li>
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                        <div class="card-content">
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    <li data-menuanchor="navigationsPage" data-hover="true" data-activates="navi-dropdown" data-gutter="-16" data-beloworigin="true" class="dropdown-button"><a href="#navigationsPage">Navigations</a></li>
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  </ul>
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                                <img src="./Overview.png" style="width:250px;">
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<ul class="dropdown-content" id="navi-dropdown">
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                            <div class="divider"></div>
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  <li><a href="https://2015.igem.org/Team:USTC/Description" class="waves-effect waves-light">Project</a></li>
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                            <h3><b id="OverviewNDM" class="scrollspy">NDM:</b><br /><b>Nanomachine Detecting Microbiotics</b></h3>
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  <li><a href="https://2015.igem.org/Team:USTC/Modeling" class="waves-effect waves-light">Modeling</a></li>
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  <li><a href="https://2015.igem.org/Team:USTC/Results" class="waves-effect waves-light">Results</a></li>
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                            <p>
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  <li><a href="https://2015.igem.org/Team:USTC/Achievements" class="waves-effect waves-light">Achievements</a></li>
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                                Abusing antibiotics has caused severe antibiotics contamination and resistance issues worldwide. Therefore, we USTC develop a device NDM with OD detector and optical interference path, recognition program based on RasberryPi and Arduino to detect antibiotics in natural water bodies.
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  <li><a href="https://2015.igem.org/Team:USTC/Measurement" class="waves-effect waves-light">Measurement</a></li>
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                                <br />
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  <li><a href="https://2015.igem.org/Team:USTC/Software" class="waves-effect waves-light">Software</a></li>
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                                <br />
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  <li><a href="https://2015.igem.org/Team:USTC/Parts" class="waves-effect waves-light">Parts</a></li>
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                                In NDM, there are two bacterial systems for measurement:
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  <li><a href="https://2015.igem.org/Team:USTC/Tutorials" class="waves-effect waves-light">Tutorials</a></li>
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                                <br />
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  <li><a href="https://2015.igem.org/Team:USTC/Notebook" class="waves-effect waves-light">Notebook</a></li>
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                                <br />
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  <li><a href="https://2015.igem.org/Team:USTC/Safety" class="waves-effect waves-light">Safety</a></li>
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                                <b id="OverviewROSE" class="scrollspy">ROSE</b>, an engineered bacterial reporter system integrated with permeability modification, logic amplification circuit and quorum sensing, is able to adjust EGFP expression level, and NDM reads fluorescence intensity from ROSE to send results in high resolution.
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  <li><a href="https://2015.igem.org/Team:USTC/Practices" class="waves-effect waves-light">Policy&Practices</a></li>
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  <li><a href="https://2015.igem.org/Team:USTC/Team" class="waves-effect waves-light">Team</a></li>
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                                <br />
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  <li><a href="https://2015.igem.org/Team:USTC/Attributions" class="waves-effect waves-light">Attributions</a></li>
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                                <b id="OverviewCACCI" class="scrollspy">CACCI</b>, chemotaxis and transmembrane protein-modified bacteria, are chemically covalently adhered to a polymer membrane. Deformation caused by CACCI motility can be recognized with optical interference and the interference pattern recognition program inside NDM will analyze the deformation, thus antibiotic situation in sample can be accurately told.
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</ul>
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                                <br /> Hope with our project, antibiotics issues can be solved in an accelerated way.
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  <div class="fullpagesection black" data-anchor="welcomePage" id="welcome">
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      <ul class="slides">
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        <li> <img src="https://static.igem.org/mediawiki/2015/2/22/USTC-index-background-1.jpg"> </li>
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                            <ul class="section table-of-contents">
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        <li> <img src="https://static.igem.org/mediawiki/2015/6/6e/USTC-index-background-2.jpg"> </li>
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        <li> <img src="https://static.igem.org/mediawiki/2015/a/a5/USTC-index-background-3.jpg"> </li>
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                                    <a href="#OverviewNDM">Overview:NDM</a>
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        <li> <img src="https://static.igem.org/mediawiki/2015/6/67/USTC-index-background-4.jpg"> </li>
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                                </li>
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    </div>
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                                    <a href="#OverviewROSE">Overview:ROSE</a>
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    <div class="container">
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                                </li>
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      <div class="check-inview fadeup">
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                                <li>
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        <h1 class="white-text"><b>Nanomachine Detecting Microbiotics</b></h1> </div>
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                                    <a href="#OverviewCACCI">Overview:CACCI</a>
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      <div class="check-inview fadeup">
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                                </li>
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        <h2>In the beginning, we extracted antibiotics, assuming it to be Perseus and it was so. Time going, we found antibiotics turned out to be Ares.</h2>
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                            </ul>
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      </div>
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                        </div>
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      <div class="check-inview fadeup center" style="text-align:center;">
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                    </div>
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        <a href="#introPage">
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                </div>
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          <h2 class="white-text">Continue</h2><img src="https://static.igem.org/mediawiki/2015/1/1d/USTC-index-Down.png" style="width: 60px;" /></a>
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      </div>
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  <div class="fullpagesection blue darken-2" data-anchor="introPage" data-delay="500" id="intro">
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    <div class="container">
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        <div class="col s12">
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          <h5 class="white-text">In natural environment, many antibiotic substances are emitted into river in their active state, which caused tremendously challenges on human health and environment. Thus, this year we USTC develop NDM, a hardware trying to detect those molecules and hold back this situation.  Advantages of NDM including: </h5>
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        </div>
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      </div>
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      <div class="row">
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        <div class="col s4 check-inview fadeup">
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          <figure><img src="https://static.igem.org/mediawiki/2015/b/b1/USTC-index-fast.png"></figure>
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          <div>
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              <h5>Rapid</h5>
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              <p>Detection won’t be based on fluorescence signal,  however a brand new way! Then, how much time does a detection assay cost? 1 day? 100 min? Answer is 100 SECONDS!</p>
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          </div>
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        </div>
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        <div class="col s4 check-inview fadeup">
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          <figure><img src="https://static.igem.org/mediawiki/2015/1/18/USTC-index-toolbox.png"></figure>
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          <div>
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              <h5>Convenient</h5>
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              <p>NDM is portable for people to operate it, and we developed a friendly GUI for better experience. Moreover, we will package bacteria into powder for iGEMers!</p>
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          </div>
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        </div>
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        <div class="col s4 check-inview fadeup">
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          <figure><img src="https://static.igem.org/mediawiki/2015/c/cd/USTC-index-gear.png"></figure>
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          <div>
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              <h5>Compatible</h5>
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              <p>NDM is compatible for everyone to engineer. We hope genetic modification on device level would expand.  After all, ‘M’ in NDM means much more than microbiotics!</p>
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          </div>
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        </div>
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      </div>
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      <div class="check-inview fadeup center" style="text-align:center;">
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        <a href="#constitutionPage">
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          <img src="https://static.igem.org/mediawiki/2015/1/1d/USTC-index-Down.png" style="width: 60px;" /></a>
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      </div>
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    </div>
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  </div>
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  <div class="fullpagesection light-blue darken-3" data-anchor="constitutionPage" id="constitution">
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    <div class="container">
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      <div class="row" style="margin-bottom: 0;">
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        <div class="col s12">
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          <h4 class="white-text">Constitutions of NDM</h4>
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        </div>
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      </div>
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      <div class="row">
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        <div class="col s2"><img src="https://static.igem.org/mediawiki/2015/c/c6/USTC-index-mod-bact.png"></div>
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        <div class="col s2"><img src="https://static.igem.org/mediawiki/2015/1/1d/USTC-index-film.png"></div>
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        <div class="col s2"><br/><img src="https://static.igem.org/mediawiki/2015/c/c4/USTC-index-hako.png" style="z-index: 5;"></div>
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        <div class="col s2"><img src="https://static.igem.org/mediawiki/2015/9/9c/USTC-index-optical-path.png"></div>
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      </div>
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      <div class="row">
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        <div class="col s2"><h5>CACCI</h5><p>Programmed bacteria to detect chemicals whatever you want</p></div>
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        <div class="col s2"><h5>Film</h5><p>Change the movement of bacterial to deformation of film.</p></div>
 +
        <div class="col s2"><h5>SPRING</h5><p>The main body NDM, platform provided for experiment.</p></div>
 +
        <div class="col s2"><h5>Optical path</h5><p>Detect the deformation by Michelson Interference.</p></div>
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        <div class="col s2"><h5>GUI</h5><p>Provide a better experience for users.</p></div>
 +
      </div>
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      <div style="text-align:center;">
 +
        <a href="#tutorialPage">
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          <img src="https://static.igem.org/mediawiki/2015/1/1d/USTC-index-Down.png" style="width: 60px;" /></a>
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      </div>
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          <h4 class="white-text">Tutorials: Study NDM Right Now!</h4>
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      <div class="row check-inview fadeup">
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        <div class="col s4 check-inview fadeup">
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          <figure>
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            <a href="https://2015.igem.org/Team:USTC/Tutorials#Instructional-Video"><img src="https://static.igem.org/mediawiki/2015/3/31/USTC-index-video.png"></a>
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          </figure>
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          <div>
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              <h5>Video</h5>
 +
              <p>Want to know how to operate NDM? Watch it at here!</p>
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          </div>
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        </div>
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        <div class="col s4 check-inview fadeup">
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          <figure>
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            <a href="https://2015.igem.org/Team:USTC/Tutorials#Instruction-manual"><img src="https://static.igem.org/mediawiki/2015/7/79/USTC-index-ins.png"></a>
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          </figure>
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          <div>
 +
              <h5>Manual</h5>
 +
              <p>This manual is a convenient guidebook for you when operating NDM in field! Download it at here!</p>
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          </div>
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            <a href="https://2015.igem.org/Team:USTC/Tutorials#Make-Your-Own"><img src="https://static.igem.org/mediawiki/2015/5/54/USTC-index-DIY.png"></a>
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          </figure>
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          <div class="card hoverable">
 +
              <h5>DIY</h5>
 +
              <p>Want to make your own NDM? Come and join us at here!</p>
 +
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                            <h3 id="what-is-antibiotics-" class="scrollspy">What is Antibiotics?</h3>
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                <span class="white-text">Modeling</span></a>
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                            <p>Antibiotics, or microbiotics, in narrow sense, are a various type of artificially constructed or naturally existing chemicals containing the capability of killing bactiera or inhibiting their growth, which are implemented widespreadly around the world. Patients who got bactieral or protozoan infection from surgical wound like war field, empyrosis and previously regarding severe diseases such as general cold, tuberculosis(TB), sexual transmitted diseases(STD), dysentery, anthrax, even cancer and so forth are able to cure treating with antibiotic substances. Since the discovery of penicillin by British biologists Alexander Flaming in 1929, the antibiotic family has been enlarged a lot. With properly administration of antibiotics, millions of people were saved in evidently alleviated symptom. There was a time when antibiotics, along with oil source for automobile, was indispensable for human beings.</p>
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                            <br />
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                            <p>Antibiotic substances containing several mechanism, rendering them to kill and inhibit bactieral growth in their own ways. Those mechanisms including:</p>
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                            <ul class="collection">
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                                <li class="collection-item">inhibit the synthesis of cell wall.</li>
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                                <li class="collection-item">influence the function of cell membrane.</li>
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                                <li class="collection-item">inhibit the biosynthesis of nucleic acid.</li>
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                                <li class="collection-item">inhibit the biosynthesis of proteins.</li>
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                            </ul>
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                            <p><img src="https://upload.wikimedia.org/wikipedia/commons/0/08/Antibiotics_Mechanisms_of_action.png" alt="From TheMedSchool">
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                                <br>Classes catogorization based on chemical structure including:</p>
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                            <ul class="collection">
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                                <li class="collection-item">Beta-lactam</li>
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                                <li class="collection-item">Aminoglycoside</li>
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                                <li class="collection-item">Amide alcohol</li>
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                                <li class="collection-item">Macrolide</li>
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                                <li class="collection-item">Polypetide</li>
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                                <li class="collection-item">Tetracycline</li>
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                                <li class="collection-item">Sulfonamides</li>
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                                <li class="collection-item">Quinolones</li>
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                                <li class="collection-item">Polymyxin</li>
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                                <li class="collection-item">Trimethprim</li>
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                                <li class="collection-item">Nitrofuran etc.</li>
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                            </ul>
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                            <p><img src="http://gardeningblogger.info/image/55572f8e4a771.jpg" alt=""></p>
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                            <p>Ordered formal course of treatment, these antibiotic would effectly treat
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                                <br>gonorrhea, MRSA and pseudomonal infections respectively.</p>
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                            <br/>
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                            <p>However, treating with microbiotics may be associated with a rang of side effects, from some mild including gastrointestinal upset, diarrhea to severe like hearing loss, kidney damage. Some speculation from scientists postulated that exposition of high concentration utilization of antibiotics alter the host microbiota, especially the familiy of the probiotics, and this has been associated with chronic diseases.</p>
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                            <br />
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                            <p>The prodcution of antibiotics in pharmceutical companies explosively expanded these years. Articifical synthesis of sulfonamides, quinolones and oxazolidinones enrich the categories of antibiotics.</p>
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                            <br />
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                            <p class="divider"></p>
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                            <br />
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                            <h3 id="antimicrobial-resistance-and-abuses" class="scrollspy">Antimicrobial Resistance and Abuses</h3>
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                            <p>As the matter of fact, microbiotics, the great saver of our life, is now putting us in potentially tremendous disaster both environmentally and physiologically. </p>
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                            <br />
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                            <p>Emergence of antimicrobial resistance is theoratically predictable by evolutionary potential. The antibiotic treatment may select for bacterial strains physiologically improved of their cell bodies or genetically enhanced capacity of mutagenesis and gene horizontal transfer like conjugation.</p>
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                            <br />
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                            <p>Because of such horizontal genetic exchange, nowadays, efficacy loss may attribute to the emergence of many antimicrobrial-resistent strains. For instance, tuberculosis(TB) in China becomes more difficult with administration with previous antibiotic substances owing to elevating level of antimicribrial resistence, which raised great challenge on Chinese public health. Besides, the discovery of NDM-1(New Delhi Metallo-beta-lactamase) in <em>Klebsiella pneumoniae</em> from a Swedish patient of Indian origin in 2008 once may contribute to severe disaster on that broad of beta-lactan antibiotics.</p>
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                            <br />
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                            <p>Though, antimicrobial resistance can be transferred through evolutionary theory, main contribution of increasing antibiotic resistance comes from individuals' inappropriate antibiotics treatment, overuse of antibiotics without prescription from professional doctor, instead from self-prescription.</p>
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                            <p><img src="http://chuantu.biz/t2/11/1438322045x-954498856.jpg" alt="图片名称"></p>
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                            <p>Demonstrably, attribution of antibiotic resistance not only from clinically, but from agriculture and animal husbandry. In China, the overuse of antibiotics for animal is the same serious as the clinical therapy. In farms or hospitals, solution containing antibiotics directly emittes to river, which potentially strongly influence the ecological equilibrium of bactierial colonies and sidelong threaten public health.</p>
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                            <br />
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                            <p class="divider"></p>
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                            <br />
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                            <h3 id="antibiotics-production-and-contamination-in-china" class="scrollspy">Antibiotics Production and Contamination in China</h3>
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                            <p>Because individuals neglecting antibiotic emission, rivers in China now encounter extremely antibiotic contamination.</p>
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                            <p><img src="image/1438323729x-1566638187.png" alt="图片名称"></p>
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                            <p>According to the latest research from <em>Chinese Academy of Science</em> published on ACS Environmental Science and Technology, China used 162,000 tons of antibiotic substances for human(48%) and animal(52%) in 2013, which is tenfold usage of USA and antibiotic substances in high concentration can be detected from many river basins that may potentially harmful to public health and environment. In their professional evaluation, antibiotics can be detected in all rivers in China in different degree. </p>
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                            <p><img src="image/1438322929x-1566638187.png" alt="图片名称"></p>
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                            <p>Among these rivers, the Pearl River Basin in South China and Hai River Basin in North China received the highest antibiotics emission. More than 36 common antibiotic substances were discovered in these rivers, one of them, for example, in the Pearl River Basin, amoxicillin was found at 3384 ng/L and fluprofen at 2867 ng/L. Though lack of standard ranking on antibiotic contamination, according to environmental scienctists from CAS, antibiotic substances concentration more than 1000 ng/L is relatively extremely high, based on Europe Union Standard.</p>
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                            <br />
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                            <p>Now, many technology including ELISA, Delvotest, HPLC-MS/MS, CE and Quantum Dot are able to detect antibiotic concentration in river sample, however much of their have much dificiencies, including:</p>
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                            <ul class="collection">
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                                <li class="collection-item">Not portable, many big and automatic equipments are indispensable for concentration detection.</li>
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                                <li class="collection-item">Complicated Routine, which requires traned personnel to conduct experiment. However, facing that large scale contamination, complicated routine and trained personnel cannot be satisfied at all districts, so do it in many developing and undeveloped countries and districts.</li>
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                                <li class="collection-item">Recent technology is still not sensitive enough and sometimes time consuming.</li>
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                            </ul>
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                            <p>Consequently, this year we iGEM constructed NDM, which is abbreviated from Nanomachine detecting microbiotics. Using NDM based on mechanism of GFP reporter, chemotaxis and interference, a portable and accurate device is established to encounter the issues today.</p>
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                            <p><a onclick="$(document).scrollTop(0)">Top</a></p>
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                            <ul class="section table-of-contents">
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                                    <a href="#what-is-antibiotics-">What is Antibiotics? </a>
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                                    <a href="#antimicrobial-resistance-and-abuses">Antimicrobial Resistance <br/>and Abuses</a>
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                                    <a href="#antibiotics-production-and-contamination-in-china">Antibiotics Production <br/>and Contamination in China</a>
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                                </li>                               
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              <a href="https://2015.igem.org/Team:USTC/Results">
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                <span class="white-text">Results</span></a>
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                            <h2 id="ROSEwhatis" class="scrollspy">What is ROSE?</h2>
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                            <p>
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                                <strong><em>Reporter Operater with Sensing Engine</em></strong>, in brief-<strong>ROSE</strong>, is a systematically modified bactieral machine for antibiotic concentration determination. Because antibiotic substances contamination occurs in very low concentration, such as ~2000ng/L or 2ug/L that is much lower than normal contaminants concentration, accurate detection by bactiera using simple genetic circuit. Consequently, ROSE is established with both genetically amplified circuit and systematic permeability reconstruction.
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                            <p class="divider"></p>
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                            <br />
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                            <h3 id="ROSESystematicOverviewofROSE" class="scrollspy">Systematic Overview of ROSE</h3>
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                            <div><img src="https://static.igem.org/mediawiki/2015/5/5b/Overview_of_ROSE.png" alt="Overview of ROSE" /></div>
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                            <p>
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                                In general, ROSE as a community, containing two kinds of bactiera, among which Bactiera I specfically receives antibiotic concentration information from <em>antibiotic substance sensing promoters</em>, which were previously selected by Shimshon Belkin and Sahar Melamed in 2012. Then Bacteria I output information with LuxI, part of genetic expression regulator by quorum sensing, whose information is provided with positive amplification for Bacteria II. Bacteria II itself will automatically produce another part of regulator LuxR triggered by IPTG. Besides Bacteria II has a logically amlification circuit regulated by LuxI-LuxR complex. In this circuit containing two promoter regulaing two protein. The former one is regulated by Lux complex and produces cI inhibition protein and the latter one will be inhibited by cI protein and EGFP will be expressed when lack of cI protein. Consequently, this genetic amplification circuit, compare with others, will not directly enhance the intensity of GFP expression but to increase the signal resolution of concentration variation.
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                            </p>
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                            <br />
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                            <p>
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                                The design intention on bactieral membrane permeability improvement is directly from part of mechanism of antibiotics resistance. Theoratically, bacteria won't receive antibiotic substance bacause of mutation of its transmembrane protein and efflux system. Thus, we are attempting to modify bactiera in reverse way, that is overexpression of transmembrane protein, or more specifically porin, and inhibition of efflux system production through CRISPR.
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                            </p>
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                            <p class="divider"></p>
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                            <br />
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                            <h3 id="ROSEAntibioticSubstanceSensingModule" class="scrollspy">Antibiotic Substance Sensing Module</h3>
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                            <p>
+
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                                Previous research from Sahar Melamed and Shimshon Belkin has already selected dozens of promoters responding to antibiotic concentration specificallly through the fluorescence signal.
+
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                            </p>
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                            <p><img src="https://static.igem.org/mediawiki/2015/7/73/Promoter_Selection_with_Fluroescence_Intensity.png" alt="Promoter Selection with Fluroescence Intensity, *2012 Shimshon Belkin* " /></p>
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                            <p>
+
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                                For instance, <em>micF</em> has relatively specific signal response when treated with sulfamethoxazole(255.2), sulfadimethoxine(44.9) and colistin(36.9), <em>soxS</em> is able to sense tetracycline(26.5) and oxytetracycline(26.1) at relative high intense. Besides, <em>recA</em> will effectively detect the existence of nalidixic acid and etc. These maximal induction of each promoter strain is tested in the course of 10h of exposure.
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                            </p>
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                            <br />
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                            <p>
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                                The mechanism of these promoters are different from each other. However, basically, mechanisms consist of: - micF, an antisense ncRNA targeting ompF RNA, main endogenous porin of <em>Escherichia coli</em>, participating in post-transcriptionally regulation of this outer membrane protein F(ompF). micF is also regarded as one of the promoter of soxRS-regulated gene.
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                                <br />
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                                <img src="https://static.igem.org/mediawiki/2015/2/22/Antisense_ncRNA_with_ompF_mRNA_5%27_UTR.png" alt="micF: antisense ncRNA with ompF mRNA 5' UTR,*2011 JMB*" />
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                                    SoxS, a transcriptional activator of oxidative stress genes in <em>E. coli</em>. SoxS <em>in vitro</em> binds the promoter of soxRS-regulated genes such as micF, sodA and recruits RNA polymerase to the promoters.
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                            <p>
+
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                                In accordance with relative effective response of antibiotics, we are determined to implement micF to sense the existence of sulfamethoxazole and sulfadimethoxine in water sample,
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                                <img src="https://static.igem.org/mediawiki/2015/a/a1/Micf.png" alt="图片名称" /></p>
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                            <p>
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                                and SoxS to detect the concentration of tetracycline or oxytetracycline.
+
−
                                <img src="https://static.igem.org/mediawiki/2015/b/b4/Soxs.png" alt="图片名称" /></p>
+
−
                            <p>In the circuit, micF and SoxS won't directly reporter fluorescence signal, instead, information will deliver through quorum sensing for positive amplification. In antibiotic substance sensing module, LuxI will be expressed directly for Bacteria II processing.</p>
+
−
                            <br />
+
−
                            <p class="divider"></p>
+
−
                            <br />
+
−
                            <h3 id="ROSEQuorumSensingModule" class="scrollspy">Quorum Sensing Module</h3>
+
−
                            <p>
+
−
                                Using community with different compartments would further improve the signal amplification, as known, quorum sensing is a system for stimulation(some signaling molecules called autoinducers) and response(receptor that will transcrpt certain genes) that is related to the bactieral concentration in the system. Many gene expression is naturally regualted by quorum sensing, which is also treated as social interation in bacterial community.
+
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                                <img src="https://static.igem.org/mediawiki/2015/0/0d/Che.png" alt="图片名称" />
+
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                            </p>
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                            <br />
+
−
                            <p>
+
−
                                In some gram-negative bacteria, such as <em>Vibro fischeri</em>, biosynthesis of autoinducers is used for yield of bioluminescence through luciferase. The signaling molecule used by <em>V. fischeri</em> is an acylated homoserine lactone(AHL),called N-(3-oxohexanol)-homoserine lactone, which can be engineered into <em>E. coli</em>. The signaling molecule will be produced in the cytoplasm using LuxI synthease enzyme and then secreted through the cell membrane to the extracelluar surroundings. When N-(3-oxohexanol)-homoserine lactone begins diffusing back into cells or other bacteria, LuxR will recognize the existence of N-(3-oxohexanol)-homoserine lactone with a threshold concentration, about 10ug/mL. Finally, target gene such as LuxlCDABE will be activated with LuxR and N-(3-oxohexanol)-homoserine lactone complex.
+
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                            </p>
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                            <br />
+
−
                            <p>
+
−
                                It has been proved that LuxR has evolutionary conservation in many gram-negative bacteria, which enables <em>E. coli</em> to receive AHL from other bacteria. Thus we engineer Bacteria I to release quorum sensing molecules to Bacteria II for exponentailly amplified results.
+
−
                            </p>
+
−
                            <br />
+
−
                            <p>
+
−
                                <img src="https://static.igem.org/mediawiki/2015/f/f3/RBS.png" alt="图片名称" />  
+
−
                                To achieve antibiotic concentration corresponding to AHL production, AHL produced by Bacteria I will be regulated by antibiotic substance sensing promoters. And in Bacteria II, LuxR will be directly expressed regulated by lac operon, triggered by IPTG. With treatment of IPTG, LuxR will be consitutively expressed to ensure LuxR-AHL complex will intiate the expression of required genes.
+
−
                            </p>
+
−
                            <br />
+
−
                            <p class="divider"></p>
+
−
                            <br />                           
+
−
                            <h3 id="ROSESecondLevelAmplificationModule" class="scrollspy">Second Level Amplification Module</h3>
+
−
                            <p>
+
−
                                In addition of signal amplification through quorum sensing, an artificial genetically engineered cascade magnification circuit is also implemented in ROSE construction.
+
−
                            </p>
+
−
                            <p>
+
−
                                <img src="https://static.igem.org/mediawiki/2015/2/25/Cl.png" alt="图片名称" />
+
−
                            </p>
+
−
                            <p>
+
−
                                In this cascade signal circuit, LuxR-AHL complex trigger the activation of promoter Lux, then express cI derived from λ phage. Since cI is a highly effcient repressor that is a low concentration of cI will completely repress the effiency of λ promoter. As a result, EGFP will be drastically and negatively regulated when treated with antibiotic substances.
+
−
                            </p>
+
−
                            <br />
+
−
                            <p>
+
−
                                It is notable that this second level amplication module won't directly expand the signal intensity, in another way, it will indirectly enlarge the resolution of different antibiotic concentration gradients. Our theoratical modeling also illustrate a higher resolution implementing this amplification circuit.
+
−
                            </p>
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                            <br />
+
−
                            <p class="divider"></p>
+
−
                            <br />                           
+
−
                            <h3 id="ROSEIntegrativeModificationonBacterialPermeability" class="scrollspy">Integrative Modification on Bacterial Permeability</h3>
+
−
                            <p>
+
−
                                Our ROSE construction is systematically not only include quorum sensing and logic amplication, but containing some permeability improvement by inhibition of efflux system and overexpression of exogenous porin. Nonpolar residues face outward so as to interact with the nonpolar lipid membrane, whereas the polar residues face inwards into the center of the beta barrel to interact with the aqueous channel.
+
−
                            </p>
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−
                            <br />
+
−
                            <p>
+
−
                                <strong>Porin: OprF from <em>Pseudomonus aeruginosa</em></strong> Porins are beta barrel proteins crossing a celluar membrane and act as a pore through which molecules are able to diffuse.
+
−
                            </p>
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−
                            <br />
+
−
                            <p>
+
−
                                In <em>E. coli</em> different porins, for example OmpF and OmpC is expressed endogenously in different situations for different molecules. But these natural occurring porines are only permeable for molecules like sugars smaller than 600 Da. And more importantly, implement of micF, an antisense ncRNA may inhibit the material diffusion through OmpF. Therefore, it is necessary to import an exogenous porin with higher effiency of small molecular tranportation, such as OprF derived from <em>Pseudomonas aeruginosa</em>.
+
−
                                <img src="https://static.igem.org/mediawiki/2015/e/ed/Pseudomonas_aeruginosa.png" alt="图片名称" />
+
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                            </p>
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−
                            <br />
+
−
                            <p>
+
−
                                OprF represents one of the largest pore sizes on bacterial outer membranes, allowing diffusion of polysaccharides in a range of 2000 to 3000 Da. The overexpression of OprF in <em>E. coli</em> will drastically improve the antibiotic absorption capability, like antibiotic condensation occured in <em>E. coli</em>.
+
−
                            </p>
+
−
                            <br />
+
−
                            <p>
+
−
                                <strong>Viroporin: SCVE from SARS Virus</strong> Viroporins are similar to porins in that they oligomerize within the membrane to form pores and this oligomerization often occurs between hydrophobic, transmembrane and alpha-helical domains of the proteins.</p>
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−
                            <p><img src="https://static.igem.org/mediawiki/2015/2/29/Proteins1.jpg" alt="图片名称" />
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                            </p>
+
−
                            <p>
+
−
                                SCVE, SARS Caronavirus Envolope protein, is a kind of effective viroporin within 76 residues that is found normally pentamerized in bacteria. SCVE is permeable for many small molecules, which is originately designed for apoptosis of cells triggered by virus. Implementation of SCVE in ROSE may potentially improve the permatilibty of small molecules including antibiotics.
+
−
                            </p>
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−
                            <br />
+
−
                            <p>
+
−
                                <strong>Inhibition of Efflux System using CRIPSR</strong> Efflux system is a kind of mechanism found in microbes responsible for many moving molecules, especially some toxic substances, microbiotics.
+
−
                                <img src="https://static.igem.org/mediawiki/2015/f/f6/Fmicb-04-00007-g001.jpg" alt="Strucutual Model of AcrAB System" />
+
−
                                <img src="https://static.igem.org/mediawiki/2015/d/d2/Strucuture_of_EmrE%2C_3B61_in_PDB.png" alt="Strucuture of EmrE, 3B61 in PDB" />
+
−
                            </p>
+
−
                            <p>
+
−
                                According to previous research, efflux system contributes a lot for bacterial antibiotic resistance, or multidrug resistance(MDR). For example, in <em>E. coli</em>, AcrAB efflux system has a physiological role of pumping out many bile acids, fatty acids to lower their intracelluar toxicty. It would be an attemptation to go directly reverse to antibiotic resistance, that is to inhibit the function of efflux system. In order to completely inhibit the major efflux system of <em>E. coli</em> in genetic level, CRIPSR would be an impressive solution for totally deletion of the function of efflux system.
+
−
                            </p>
+
−
                            <br />
+
−
                            <p>
+
−
                                Clutersted reularly interspacaed short palindromic repeats, abbreviated as <em>CRIPSR</em>, are some fragements in prokayotic DNA with short repeats. Natually, CRISPR/Cas system is an important immune system that is able to resist the existence and reproduction of exogenous genetic materials, directly recognizing and then cut these genetic fragments, such as phages and plasmids, by CRIPSR spacers, which is quite similar to RNA interference(RNAi) discovered in eukaryotic organisms. Cas9, which is CRISPR Associated Protein 9 in brief, is an RNA-guding DNA endonuclease enzyme associated with the CRISPR segments derived naturally from * Streptococcus pyogenes* and other bacteria. It has been showed that Cas9 plays an important role in memorizing, interrogating and ultimately cleaving these foreign DNA. Mechanism of cleavage is to unwind the foreign DNA and check whether there is a about 20 bp fragments that is complementary to guide RNA (gRNA, in brief). When the recognition result turns to yes, Cas9 will cleave the foreign DNA. Nowadays, with proper modification, CRIPSR/Cas9 has become a common gene editing tool, being able to cut the target gene with gRNA design.
+
−
                            </p>
+
−
                            <p><img src="https://static.igem.org/mediawiki/2015/9/9b/Crispr-caslg.jpg" alt="Schematic Model of CRISPR/Cas9 System" /></p>
+
−
                            <p>
+
−
                                When it comes to artificial design of CRISPR/Cas9 system, how to sucessfully achieve recruitment of Cas9 depends on the structure of gRNA. In general, gRNA consists of 2 main parts, the former part is about 20 bp RNA sequences, which is exactly complementary to target genes, and it will be more impressive if these complementary sequence is at the beginning of the transcrption sites of gene. And the latter part is a secondary structure part which finally forms as hairpin used to recruit Cas9. During engineered section, this structual part can directly import from iGEM parts and what we need to take care is the fragment complementary to efflux system gene.
+
−
                            </p>
+
−
                            <p>CRISPR/Cas9 System for AcrB:
+
−
                                <img src="https://static.igem.org/mediawiki/2015/4/49/CRISPR_Cas9_System_for_AcrB.png" alt="图片名称" /></p>
+
−
                            <p>CRISPR/Cas9 System for EmrE:
+
−
                                <img src="https://static.igem.org/mediawiki/2015/f/fc/CRISPR_Cas9_System_for_EmrE.png" alt="图片名称" /></p>
+
−
                            <br />
+
−
                            <p class="divider"></p>
+
−
                            <br />
+
−
                            <h3 id="ROSETheoreticalAdvantage" class="scrollspy">Theoretical Advantage</h3>
+
−
                            <p><img src="image/QQ%E6%88%AA%E5%9B%BE20150803214403.png" alt="图片名称" />
+
−
                                <img src="image/QQ%E6%88%AA%E5%9B%BE20150803214410.png" alt="图片名称" /></p>
+
−
                            <br />
+
−
                            <p class="divider"></p>
+
−
                            <br />
+
−
                            <h3 id="ROSEBibliography" class="scrollspy">Bibliography</h3>
+
−
                            <p>
+
−
                                1.<i>A bacterial reporter panel for the detection and classification of antibiotic substances <b>Microbial Biotechnology (2012) 5(4), 536-548</b>Sahar Melamed, Shimshon Belkin</i>
+
−
                            </p>
+
−
                            <br />
+
−
                            <p>
+
−
                                2.<i>Signal-Amplifying Genetic Circuit Enables In Vivo Observation of WeakPromoter Activation in the Rhl Quorum Sensing System<b>Wiley InterScience 2005</b>DOI: 10.1002/bit.20371</i>
+
−
                            </p>
+
−
                            <br />
+
−
                            <p>
+
−
                                3.<i>MicF: An Antisense RNA Gene Involved in Response of Escherichia coli to Global Stress Factors <strong>2001 JMB</strong> doi:10.1006/jmbi.2001.5029</i>
+
−
                            </p>
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−
                        </div>
+
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                    </div>
+
−
                    <div class="col hide-on-small-only m3">
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                        <div class="toc-wrapper pinned">
+
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                            <ul class="section table-of-contents">
+
−
                                <li>
+
−
                                    <a href="#ROSEwhatis">What is ROSE?</a>
+
−
                                </li><div class="divider"></div>
+
−
                                <li>
+
−
                                    <a href="#ROSESystematicOverviewofROSE">Systematic Overview <br/>of ROSE</a>
+
−
                                </li><div class="divider"></div>
+
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                                <li>
+
−
                                    <a href="#ROSEAntibioticSubstanceSensingModule">Antibiotic Substance<br/> Sensing Module</a>
+
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                                </li><div class="divider"></div>
+
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                                <li>
+
−
                                    <a href="#ROSEQuorumSensingModule">Quorum Sensing Module</a>
+
−
                                </li><div class="divider"></div>
+
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                                <li>
+
−
                                    <a href="#ROSESecondLevelAmplificationModule">Second Level<br/> Amplification Module</a>
+
−
                                </li><div class="divider"></div>
+
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                                <li>
+
−
                                    <a href="#ROSEIntegrativeModificationonBacterialPermeability">Integrative Modification <br/>on Bacterial Permeability</a>
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                                </li><div class="divider"></div>
+
−
                                <li>
+
−
                                    <a href="#ROSETheoreticalAdvantage">Theoretical Advantage</a>
+
−
                                </li><div class="divider"></div>
+
−
                                <li>
+
−
                                    <a href="#ROSEBibliography">Bibliography</a>
+
−
                                </li>
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+
−
                            </ul>
+
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                        </div>
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                    </div>
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                </div>
+
 
             </div>
 
             </div>
 +
          </div>
 
         </div>
 
         </div>
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         <div id="CACCI" class="row">
+
         <div class="col s3 navi-inview fadeup" id="nav4">
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            <div class="card hoverable">
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          <div class="card hoverable">
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                <div class="row">
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            <div class="card-image">
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                    <div class="col s12 m9">
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              <a href="https://2015.igem.org/Team:USTC/Tutorials">
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                        <div class="card-content">
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                <img src="https://static.igem.org/mediawiki/2015/d/d5/USTC-index-Tutorials.png">
−
                            <h3 id="who-is-cacci-" class="scrollspy">Who is CACCI?</h3>
+
                <span class="white-text">Tutorials</span></a>
−
                            <p>
+
−
                                <strong><em>CACCI</em></strong>, called the same as Kathy, is a constructed bacteria for <strong><em>Characterization of Antibiotic Concentration based on Chemotaxis and Interference.</em></strong>
+
−
                            </p>
+
−
                            <br />
+
−
                            <p>Although, construction of ROSE will specifically recognize the concentration of different antbiotics in the river samples, its feature is also deficiency: that is too specificity may hurt extensive measurement. Besides, using traditional genetically modified circuit to express report is still time-consuming, comparing with general physical or chemical measurement approaches.</p>
+
−
                            <br />
+
−
                            <p>Consequently, we orginally esatblished CACCI based on chemotaxis and interference to make a wider scale level antibiotic molecules detection, even other chemical molecules. So, the mechanism and construction of CACCI can be divided into the following schedules:</p>
+
−
                            <br />
+
−
                            <ol>
+
−
                                <li><strong>Chemotaxis Modification</strong>: to increase the bacterial movement capability, modification based on <em>E. coli</em> chemotaxis related parts cheZ will be introduced into bacteria.</li>
+
−
                                <li><strong>Adhesion</strong>: According to our modeling, we need to adhere bacteria to a surface. Theoratically, interference will be observed when elastic deformation occured on membrane caused by bacteria motility containing cheZ parts, covalent ligation will be established between bacteria and membrane to ensure the stable adhesion. In order to achieve covalently adhesion, modification on bacteria transmembrane protein will be necessary.</li>
+
−
                                <li><strong>Interference</strong>: When bacteria have been adhered to membrane and treated in water sample, movement will cause interference. The interference pattern will be different at distinct chemical molecules concentration.</li>
+
−
                                <li><strong>Processing</strong>: These patterns will be captured by CCD camera. Our control system containing interference pattern recognition will process these patterns, and finally a work curve will be established corresponding antibiotics concentration to different interference pattern. For friendly operation, graphic user interface(GUI) is established better for user utilization on this program.</li>
+
−
                            </ol>
+
−
                            <br />
+
−
                            <p class="divider"></p>
+
−
                            <br />
+
−
                            <h3 id="chemotaxis-modification-module" class="scrollspy">Chemotaxis Modification Module</h3>
+
−
                            <p><strong>What is Chemotaxis?</strong>
+
−
                                <br />
+
−
                                Chemotaxis is a phenomenon of the movement of an organism, specifically cells or bacteria, stimulated by chemical molecules. As for bacteria, naturally, chemoreceptors were expressed on bacteria membrane, which enables bacteria to 'sense' the existence of chemical molecule concentration in the surroundings. Chemotaxis consists of two situation, including positive chemotaxis and negative chemotaxis, which respectively demonstrate different behaviour when exposed to surroundings with chemical molecules beneficial or harmful to bactiera metabolism and reproduction.
+
−
                            </p>
+
−
                            <br />
+
−
                            <p>The chemotaxis mechanism in <em>E. coli</em> can be illstustrated as following:</p>
+
−
                            <p><img src="image/Chemotaxis.PNG" alt="Mechanism of Chemotaxis, from WITS-CSIR_SA 2011 iGEM"></p>
+
−
                            <ul>
+
−
                                <li>
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                                    <p>When absence of chemical, there is no molecule binds to chemorecptor, cheW, an intracellular protein, is associated with cheA and cheA will autophosphorylate itself and capture phosphate group from ATP. Later, phosphate will be transferred to cheY and the phosphorylated cheY associates with the flagellar motors making flagellum rotates clockwise. In general, bacteria tumble in the absence of chemical.</p>
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                                </li>
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                                <li>
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                                    <p>When presence of chemicals, cheA is free from cheW interation, and cheZ will efficiently remove phosphate group from cheY. Those dephosphorylated cheY cannot bind with flagellar motors, and consequently, flagellar rotats counter-clock which can be observed as bacteria swim directly. In some special situation, cheR will methylates chemoreceptors to remain the disasscoation between cheA and cheW. Then, bacteria will still swim without tumbles.</p>
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                                </li>
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                            </ul>
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                            <p><strong>Genetically Engineered Chemotaxis Bacteria</strong>
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                                <br>Because of lack of natural chemoceptor for antibiotics, we need to construct a genetic circuit specifically to sensing antibiotics, and according to the mechanism of chemotaxis, we need to overexpress cheZ to improve its phosphorlation capability on cheY. The model is illstruated as following:
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                            </p>
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                            <br />
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                            <p class="divider"></p>
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                            <h3 id="adhesion-module" class="scrollspy">Adhesion Module</h3>
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                            <p><strong>What Material we Implement?-Theoratical Analysis</strong>
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                                <br>In order to produce slight deformation of polymer membrane, we need to find a proper membrane to form this phenomenon. According to our modeling, see more in <a href="http://tower.im">Modeling-Determination of Polyer Membrane Candidate</a>, pressure caused by adhesive membrane is about 10^-4 Pa. Consequently, the wavelength of laser about 650nm is able to detect um-scale deformation and considering cm sqaured film, we need the Young's Modulus of membrane is smaller than 1GPa.
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                                <br><img src="image/QQ%E6%88%AA%E5%9B%BE20150810210009.png" alt="图片名称"></p>
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                            <p>The Young's Modulus of polyproylane is about 1.5Gpa, so it is selected as our candidate polymer membrane.</p>
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                            <p><strong>How to Achieve Stable Adhesion?</strong>
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                                <br>Bacterial adhereing to polymer membrane is one of the most important part in CACCI.In order to make bacteria binding tightly, chemically covalent bind of bacteria with polymer membrane maybe promising.</p>
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                            <p>So we decided to implement the parts from <em>2014 iGEM TU_Eindhoven</em>, and the whole system including these parts:</p>
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                            <ul>
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                                <li>
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                                    <p>DBCO-PEG4-NHS Ester (Dibenocyclooctyne-Polyethylene glycol-N-Hydroxysuccinimide), which covalently binding to polymer membrane.
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                                        <br><img src="image/1437880035x-954497725.png" alt="图片名称"></p>
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                                </li>
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                                <li>
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                                    <p>pAzF(p-Azido-L-Phenylalanine), a rare amino acid that would be implemented in bacterial transmembrane protein biosynthesis.
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                                        <br><img src="image/1437880130x-954497725.png" alt="图片名称"></p>
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                                </li>
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                                <li>
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                                    <p>In order to introduce pAzF into bacterial transmembrane protein, we need to mutate one of transmembrane protein, which is called COMPX. COMPX is able to form mutatedly with a special tRNA. The plasmid containing tRNA as following:
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                                        <br><img src="image/1436688284x-1376436670.png" alt="图片名称"></p>
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                                </li>
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                            </ul>
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                            <ul>
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                                <li>Covalent binding between DBCO and pAzF goes as following:
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                                    <br><img src="image/1436688789x-1376436664.png" alt=""></li>
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                            </ul>
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                            <p>To see more about constitution of our adhesion system, please refer to <a href="http://tower.im">Notebook-Protocol-Adhesion Preparation</a></p>
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                            <p><strong>Mutation of Transmembrane Protein OMPx in <em>E. coli</em></strong>
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                                <br>OMPx is a intergral outer membrane protein X from <em>Escherichia coli</em>, belonging to a family of highly conserved bacterial protein that promote bacterial adhesion to and entry into mamalian cells. Besides, these proteins as well play an important role in the resistance against attack by human complement system.
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                                <br> The modified COMPX structure is illustrated as below:
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                                <br><img src="image/1437880487x-954497725.png" alt="">
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                                <br> As planned, OMPx will be mutated at residue 16, from glycine to p-Azido-L-Phenylalanine, zooming in the mutated site in orange:
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                                <br><img src="image/1436688800x-1376436664.png" alt=""></p>
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                            <p><strong>The Covalent Reaction Model</strong>
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                                <br>Consequently, the model of adhesion goes like this: modifed bacterial containing special p-Azido-L-Phenylalanine transformed tRNA carry OMPx with mutated p-Azido-L-Phenylalanine site. The laminated side in pAzF will covalently bind to alkynyl in DBCO-PEG4-NHS ester, and consequently form chemical reaction.
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                                <br><img src="image/1437880652x-954497725.png" alt="图片名称"></p>
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                            <br />
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                            <p class="divider"></p>
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                            <br />
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                            <h3 id="optical-interference-module" class="scrollspy">Optical Interference Module</h3>
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                            <p><strong>What is Optical Interference?</strong>
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                                <br>Interference, in general physics, is a phenomenon when two waves superpose to form a resultant wave with a greater or lower amplitude, respectively called as constructive interference and destructive interference.
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                                <br><img src="image/QQ%E6%88%AA%E5%9B%BE20150810214624.png" alt="Mechiansm of Genereal Interference">
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                                <br>As for optical interference, because of high frequency of light waves(about 10^14 HZ), is it impossible to detect using current detector. Instead, only the intensity of an optical interference patteren can be observed, which is proportional to the square of the average amplitude of the wave.
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                                <br><img src="image/3800a8e0fb37906a8c6e138d105e2010.png" alt="图片名称">
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                                <br>The requirements of light source should be monochromatic, which means waves have a single frequency. Generally, a laser beam, in reality, approximates more closely to a monochromatic source and its straightforward generates interference
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                                <br><strong>Michelson interferometer</strong>
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                                <br><strong>Construction of NDM</strong></p>
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                            <br />
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                            <h3 id="processing-module" class="scrollspy">Processing Module</h3>
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                            <p><strong>Working Curve Construction</strong>
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                                <br><strong>GUI</strong></p>
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                            <ul class="section table-of-contents">
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                                    <a href="#who-is-cacci-">Who is CACCI?</a>
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                                <div class="divider"></div>
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                                    <a href="#chemotaxis-modification-module">Chemotaxis Modification <br/>Module</a>
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                                <div class="divider"></div>
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                                    <a href="#adhesion-module">Adhesion Module</a>
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                                <div class="divider"></div>
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                                <li>
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                                    <a href="#optical-interference-module">Optical Interference <br />Module</a>
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                                <div class="divider"></div>
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                                    <a href="#processing-module">Processing Module</a>
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Latest revision as of 02:58, 19 September 2015