Difference between revisions of "Team:OUC-China/InterLab"

 
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{{OUC-China}}
 
{{OUC-China}}
 
{{OUC-China/CSS}}
 
{{OUC-China/CSS}}
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<html>
 
<html>
 
<head>
 
<head>
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<title>InterLab</title>
 
<title>InterLab</title>
 
<style type="text/css">
 
<style type="text/css">
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body{padding-top: 50px;}
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.ouc-right{position: relative;}
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.ouc-navbar{background-color:#008EA1;}
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#ouc-logo{padding-top:5px;}
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.ouc-right:hover .ouc-rightnav{display: inherit;}
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#ouc-logo:hover{background-color:#008EA1;}
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.ouc-rightnav a{background-color: white; color: #008EA1; width: 100%;height: 40px; line-height: 40px;}
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.ouc-rightnav a:visited {color: #008EA1;text-decoration: none; }
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.ouc-rightnav a:hover {background-color:#D2D2D2; color: white;text-decoration: none; }
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.ouc-vicedown a{display: block;height: 40px; width: 100%;text-decoration: none;color: #008EA1; line-height: 40px;text-align: center;}
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.ouc-vicedown a:visited{color:#008EA1;text-decoration: none }
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.ouc-vicedown a:hover{color:  #EDEC8Cl}
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 +
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.ouc-nav{color:white; background-color: #008EA1;display:block; width: 100%; color: white;padding: 0}
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.ouc-nav:visited{text-decoration: none; color: white}
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.ouc-nav:hover{text-decoration: none; background-color: white; color:#008EA1; }
 +
.ouc-navbar{position: relative;padding: 0;margin: 0;}
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.ouc-navbar:hover .ouc-down{display: inherit;}
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.ouc-down{position: absolute; top:50px;display: none;background-color: white; z-index: 1001}
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.ouc-navdown{color: #008EA1;display: block; width: 100%;height: 40px;}
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.ouc-navdown:hover{background-color:#D2D2D2;color: white;text-decoration: none; z-index: 1501}
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.ouc-guide{position: fixed; width: 100%;top: 15px;z-index:1000}
 
.ouc-page-header{border-bottom-color: #EDEC8C;border-bottom-width: thick;color: #008F75;}
 
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}
 
.ouc-reserve{background-color: #008EA1;color: white;}
 
.ouc-reserve{background-color: #008EA1;color: white;}
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.ouc-heading{height:40px;border-left-width:10px;border-left-color:#E3D434; }
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    .ouc-heading {margin: 0;color: #008EA1;}
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.ouc-heading h3{margin: 0;color: #008EA1;}
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.ouc-a{color:#E3D434; }
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.ouc-ul{border-left:thin dashed #008F75}
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</style>
 
</style>
 
</head>
 
</head>
  
 
<body>
 
<body>
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<!--此处是导航-->
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<div class="ouc-guide" style="height: 50px;background-color:#008EA1 ;">
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<div class="row">
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<div class="col-md-2 navbar-header" style="text-align: center; padding: 0">
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<button class="navbar-toggle" type="button" data-toggle="collapse" data-target=".navbar-responsive-collapse">
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        <span class="sr-only">Toggle Navigation</span>
 +
        <span class="caret" style="color: white"></span>
  
−
 
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      </button>
−
<div class="navbar navbar-fixed-top ouc-navbar" role="navigation">
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<a href="https://2017.igem.org/Team:OUC-China"><img src="https://static.igem.org/mediawiki/2017/8/89/T--OUC-China--logo.jpg" height="50px"/></a></div>
−
    <ul class="nav navbar-nav">
+
−
        <li><a href="#" class="navbar-brand" id="ouc-logo"><img src="../T--OUC-China--logo.jpg" width="66" height="40" alt=""/></a></li>
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−
        <li class="dropdown">
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<div class="collapse navbar-collapse navbar-responsive-collapse" style="padding: 0">
−
            <a href="#" data-toggle="dropdown" class="dropdown-toggle ouc-nav-a">Team<span class="caret"></span></a>
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−
            <ul class="dropdown-menu">
+
−
                <li><a href="#">Member</a></li>
+
−
                <li><a href="#">Collaborations</a></li>
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<div class="col-md-1" style="padding: 0"></div>
−
                <li><a href="#">Attributions</a></li>
+
<div class="col-md-1 ouc-navbar" style="padding: 0">
−
            </ul>
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    <a href="##" style="line-height: 50px;text-align: center" class="ouc-nav">Team<span class="caret"></span></a>
−
        </li>
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    <div class="ouc-down" style=" width: 100%; text-align: center">
−
        <li class="dropdown">
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    <div><a href="https://2017.igem.org/Team:OUC-China/Team" style="line-height: 40px; " class="ouc-navdown">Members</a></div>
−
            <a href="#" data-toggle="dropdown" class="dropdown-toggle ouc-nav-a">Project<spam class="caret"></span></a>
+
    <div><a href="https://2017.igem.org/Team:OUC-China/Collaborations" style="line-height: 40px; " class="ouc-navdown">Collaborations</a></div>
−
            <ul class="dropdown-menu">
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    <div><a href="https://2017.igem.org/Team:OUC-China/Attributions" style="line-height: 40px; " class="ouc-navdown">Attributions</a></div>
−
                <li><a href="#">Description</a></li>
+
    </div>
−
                <li><a href="#">Design</a></li>
+
</div>
−
                 <li><a href="#">Experiments</a></li>
+
<div class="col-md-1 ouc-navbar" style="padding: 0">
−
                <li><a href="#">Results</a></li>
+
    <a href="##" style="line-height: 50px;text-align: center" class="ouc-nav">Project<span class="caret"></span></a>
−
                <li  class="active"><a href="#">Interlab</a></li>
+
    <div class="ouc-down" style=" width: 100%; text-align: center">
−
                <li><a href="#" >Notebook</a></li>
+
    <div><a href="https://2017.igem.org/Team:OUC-China/Description" style="line-height: 40px; " class="ouc-navdown">Description</a></div>
−
            </ul>
+
    <div><a href="https://2017.igem.org/Team:OUC-China/Design" style="line-height: 40px; " class="ouc-navdown">Design</a></div>
−
        </li>
+
                 <div class="ouc-right">
−
        <li><a href="#" class="ouc-nav-a">Model</a></li>
+
        <a style="line-height: 40px; font-size: 15px;" class="ouc-navdown" href="https://2017.igem.org/Team:OUC-China/proof1">Proof of concept</a>
−
        <li><a href="#" class="ouc-nav-a">Parts</a></li>
+
        <div class="ouc-rightnav">
−
        <li><a href="#" class="ouc-nav-a">Safety</a></li>
+
        <a href="https://2017.igem.org/Team:OUC-China/proof1" style="display: block">Basic fermentation</a>
−
        <li><a href="#" class="ouc-nav-a">Human Practice</a></li>
+
        <a href="https://2017.igem.org/Team:OUC-China/proof2" style="display: block">Adhesion platform</a>
−
    </ul>
+
        <a href="https://2017.igem.org/Team:OUC-China/proof3" style="display: block">MINI-GRE</a>
−
</div>
+
        </div>
−
 
+
    </div>
 +
    <div><a href="https://2017.igem.org/Team:OUC-China/Demonstrate" style="line-height: 40px; " class="ouc-navdown">Demonstration</a></div>
 +
    <div><a href="https://2017.igem.org/Team:OUC-China/InterLab" style="line-height: 40px; " class="ouc-navdown">InterLab</a></div>
 +
    <div><a href="https://2017.igem.org/Team:OUC-China/Improve" style="line-height: 40px; " class="ouc-navdown">Improvement</a></div>
 +
    <div><a href="https://2017.igem.org/Team:OUC-China/Notebook" style="line-height: 40px; " class="ouc-navdown">Notebook</a></div>
 +
    </div>
 +
</div>
 +
<div class="col-md-1" style="padding: 0"><a href="https://2017.igem.org/Team:OUC-China/Model" style="line-height: 50px;text-align: center" class="ouc-nav">Model</a></div>
 +
<div class="col-md-1" style="padding: 0"><a href="https://2017.igem.org/Team:OUC-China/Parts" style="line-height: 50px;text-align: center" class="ouc-nav">Parts</a></div>
 +
<div class="col-md-1" style="padding: 0"><a href="https://2017.igem.org/Team:OUC-China/Safety" style="line-height: 50px;text-align: center" class="ouc-nav">Safety</a></div>
 +
<div class="col-md-2 ouc-navbar" style="padding: 0">
 +
    <a href="https://2017.igem.org/Team:OUC-China/Engagement" style="line-height: 50px;text-align: center" class="ouc-nav">Human Practice<span class="caret"></span></a>
 +
    <div class="ouc-down" style=" width: 100%; text-align: center">
 +
    <div><a href="https://2017.igem.org/Team:OUC-China/HumanPractice" style="line-height: 40px; " class="ouc-navdown">Overview</a></div>
 +
    <div><a href="https://2017.igem.org/Team:OUC-China/HP/Silver" style="line-height: 40px; " class="ouc-navdown">Silver</a></div>
 +
    <div><a href="https://2017.igem.org/Team:OUC-China/HP/Gold_Integrated" style="line-height: 40px; " class="ouc-navdown">Gold and integrated</a></div>
 +
    <div><a href="https://2017.igem.org/Team:OUC-China/Engagement" style="line-height: 40px; " class="ouc-navdown">Engagement</a></div>
 +
    </div>
 +
</div>
 +
<div class="col-md-2" style="padding: 0"></div>
 +
 +
 +
</div>
 +
 +
 +
 +
 +
</div>
 +
</div>
 +
<!--此处是导航-->
  
 
<div class="container">
 
<div class="container">
 
     <div class="page-header ouc-page-header">
 
     <div class="page-header ouc-page-header">
−
         <h1><strong>Interlab</strong></h1>
+
         <h1 id="background"><strong>Interlab</strong></h1>
 
     </div>
 
     </div>
  
  
−
    <div class="row">
 
−
        <div class="col-md-10 ">
 
−
            <blockquote class="ouc-heading"><h3 id="introduction">Introduction</h3></blockquote>
 
−
            <p>For us, this is the second time to join it.And we test 8 circuits as the HQ told us.</p>
 
  
 +
           
 +
            <h2 class="ouc-heading"><strong>Background</strong></h2>
 +
            <p id="design" style="font-size:20px;">
 +
                    Fluorescence data can not be directly compared, it is difficult to repeat the measurement. Different instruments, different settings and different measurement environments make this difficult. Through the Interlab study, we expect to have uniform fluorescence measurement steps and standards. These protocols solve this problem by establishing absolute units on the plate reader.
 +
                </p>
 +
               
 +
                <h2 class="ouc-heading"><strong>Design</strong></h2>
 +
                <p style="font-size:20px;">
 +
                <strong>OD 600 Reference point</strong><br/>
 +
                    Using LUDOX-S40 as a reference, the ratiometric conversion factor of Abs and OD600 can be obtained.<br/>
 +
                    <strong id="materials">Protocol fluorescein fluorescence standard curve</strong><br/>
 +
                    By means of the gradient of the fluorescence standard curve, a method of obtaining a fluorescence correction of the cell can be obtained.<br/>
 +
                    <strong>Cell measurement</strong><br/>
 +
                    At four time points, the fluorescence intensity of the eight circuits was measured.
  
−
            <blockquote class="ouc-heading"><h3 id="background">Background</h3></blockquote>
 
−
            <p>
 
−
                    Measured data from the world can help us recognize how repetitive fluorescence measurements
 
−
                    are under the same protocol.As the most commonly used marker, the fluorescent measurement of
 
−
                    green fluorescent protein develops a robust measurement through interlab.
 
 
                 </p>
 
                 </p>
  
−
 
+
             <h2 class="ouc-heading"><strong>Materials and Method</strong></h2>
−
             <blockquote class="ouc-heading"><h3 id="materials">Materials</h3></blockquote>
+
             <p style="font-size:20px;">
−
             <p>
+
             <strong>Plasmids used</strong><br/>
−
             <strong>plasmids used</strong><br/>
+
             Kit Plate 6 InterLab Part Locations<br/>
−
             we get it from kit plate 6:<br/>
+
                     Positive Control (<a href="http://parts.igem.org/Part:BBa_I20270">BBa_I20270</a>): well 20B<br/>
−
                     Positive Control (BBa_I20270): well 20B<br/>
+
                     Negative Control (<a href="http://parts.igem.org/Part:BBa_R0040">BBa_R0040</a>): well 20D<br/>
−
                     Negative Control (BBa_R0040): well 20D<br/>
+
                     Test Device 1(<a href="http://parts.igem.org/Part:BBa_J364000">BBa_J364000</a>): well 20F<br/>
−
                     Test Device 1 (BBa_J364000): well 20F<br/>
+
                     Test Device 2(<a href="http://parts.igem.org/Part:BBa_J364001">BBa_J364001</a>): well 20H<br/>
−
                     Test Device 2 (BBa_J364001): well 20H<br/>
+
                     Test Device 3(<a href="http://parts.igem.org/Part:BBa_J364002">BBa_J364002</a>): well 20J<br/>
−
                     Test Device 3 (BBa_J364002): well 20J<br/>
+
                     Test Device 4(<a href="http://parts.igem.org/Part:BBa_J364003">BBa_J364003</a>): well 20L<br/>
−
                     Test Device 4 (BBa_J364003): well 20L<br/>
+
                     Test Device 5(<a href="http://parts.igem.org/Part:BBa_J364004">BBa_J364004</a>): well 20N<br/>
−
                     Test Device 5 (BBa_J364004): well 20N<br/>
+
                     Test Device 6(<a href="http://parts.igem.org/Part:BBa_J364005">BBa_J364005</a>): well 20P<br/>
−
                     Test Device 6 (BBa_J364005): well 20P<br/>
+
             <strong>Strain</strong><br/>
−
             <strong>strain used</strong><br/>
+
             <i>E.coli</i> Competent DH5α （ TaKaRa Code : D9057）<br/>
−
             E.coli Competent Cells DH5α （ TaKaRa Code : D9057）<br/>
+
             <strong>Materials</strong><br/>
−
             <strong>machines</strong><br/>
+
            1ml LUDOX (provided in kit) H20<br/>
 +
                    Fluorescein (provided in kit) 10ml 1xPBS96 well plate, black with flat bottom<br/>
 +
                    LB (Luria Bertani) media Chloramphenicol<br/>
 +
                    50 ml Falcon tube Incubator at 37°C 1.5 ml eppendorf tubes for sample storage Ice bucket with ice Pipettes<br/>
 +
            <strong id="protocol">Machines</strong><br/>
 
             Tecan Spark™ 10M<br/>
 
             Tecan Spark™ 10M<br/>
−
             <strong>software</strong><br/>
+
             <strong>Software</strong><br/>
 
             Microsoft Office EXCEL
 
             Microsoft Office EXCEL
 
             </p>
 
             </p>
  
  
−
             <blockquote class="ouc-heading"><h3 id="protocol">Protocol</h3></blockquote>
+
             <h2  id="results" class="ouc-heading"><strong>Protocol</strong></h2>
 +
          <a href="https://static.igem.org/mediawiki/2017/8/85/InterLab_2017_Plate_Reader_Protocol.pdf">InterLab_2017_Plate_Reader_Protocol</a>
  
 +
            <h2 class="ouc-heading"><strong>Results</strong></h2>
 +
<p style="font-size:20px;">
 +
                    <strong>OD 600 Reference point</strong><br/>
 +
</p>
 +
                    <table class="table">
 +
                       
 +
                        <thead>
 +
                       
 +
                        <tr>
 +
                        <th>&nbsp;</th><th>LUDOX-HS40</th><th>H<sub>2</sub>O</th>
 +
                        </tr>
 +
                        </thead>
 +
                        <tbody>
 +
                        <tr>
 +
                        <td>Replicate 1</td><td>0.0497</td><td>0.0422</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Replicate 2</td><td>0.0518</td><td>0.0495</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Replicate 3</td><td>0.0546</td><td>0.0428</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Replicate 4</td><td>0.0521</td><td>0.0569</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Arith. Mean</td><td>0.05205</td><td>0.04785</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Corrected Abs600</td><td>0.0042</td><td>&nbsp;</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Reference OD600</td><td>0.0425</td><td>&nbsp;</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>OD600/Abs600</td><td>10.1190476</td><td>&nbsp;</td>
 +
                        </tr>
 +
                        </tbody>
 +
                       
 +
            </table>
 +
                    <p style="color: gray;text-align: center;">Table 1. OD600 Reference Point</p>
 +
                    <br/>
 +
                    <p style="font-size:20px;"><strong>Protocol fluorescein fluorescence standard curve</strong></p><br/>
 +
                    <p style="color: gray;text-align: center;">
 +
                        <img src="https://static.igem.org/mediawiki/2017/8/8e/T--OUC-China--F.S.C_Fluorescein.Standard.Curve.png"><br/>
 +
                        Figure 1.Fluorescein fluorescence standard curve
 +
                    </p>
 +
                    <br/>
 +
                    <p style="font-size:20px;"><strong>Cell measurement</strong></p><br/>
 +
                    <table class="table">
 +
                    <thead>
 +
                    <tr>
 +
                    <th>&nbsp;</th><th colspan="2" style="text-align: center;">0h</th><th colspan="2" style="text-align: center;">2h</th><th colspan="2" style="text-align: center;">4h</th><th colspan="2" style="text-align: center;">6h</th>
 +
                    </tr>
 +
                    </thead>
 +
                        <tbody>
 +
                        <tr>
 +
                        <th>&nbsp;</th><th>Abs600</th><th>FI</th><th>Abs601</th><th>FI</th><th>Abs602</th><th>FI</th><th>Abs603</th><th>FI</th>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Negative Control (Colony 1)</td><td>0.0301</td><td>7</td><td>0.1156</td><td>8</td><td>0.5106</td><td>14</td><td>0.7453</td><td>17</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Negative Control (Colony 2)</td><td>0.0408</td><td>5</td><td>0.1352</td><td>8</td><td>0.5184</td><td>17</td><td>0.7326</td><td>16</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Positive Control (Colony 1)</td><td>0.0308</td><td>28</td><td>0.0805</td><td>65</td><td>0.4715</td><td>476</td><td>0.7293</td><td>722</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Positive Control (Colony 2)</td><td>0.0437</td><td>31</td><td>0.1498</td><td>136</td><td>0.4956</td><td>428</td><td>0.7049</td><td>677</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Test Device 1: J23101.BCD2.E0040.B0015 (Colony 1)</td><td>0.0283</td><td>46</td><td>0.0883</td><td>152</td><td>0.4358</td><td>606</td><td>0.6846</td><td>948</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Test Device 1: J23101.BCD2.E0040.B0015 (Colony 2)</td><td>0.0261</td><td>38</td><td>0.0969</td><td>119</td><td>0.3719</td><td>464</td><td>0.5960</td><td>738</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Test Device 2: J23106.BCD2.E0040.B0015 (Colony 1)</td><td>0.0378</td><td>44</td><td>0.1120</td><td>164</td><td>0.5090</td><td>718</td><td>0.7145</td><td>1258</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Test Device 2: J23106.BCD2.E0040.B0015 (Colony 2)</td><td>0.0223</td><td>16</td><td>0.0788</td><td>115</td><td>0.4045</td><td>506</td><td>0.5945</td><td>963</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Test Device 3: J23117.BCD2.E0040.B0015 (Colony 1)</td><td>0.0323</td><td>1</td><td>0.0945</td><td>10</td><td>0.4482</td><td>22</td><td>0.5704</td><td>28</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Test Device 3: J23117.BCD2.E0040.B0015 (Colony 2)</td><td>0.0156</td><td>4</td><td>0.0537</td><td>8</td><td>0.3399</td><td>18</td><td>0.5254</td><td>24</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Test Device 4: J23101+I13504 (Colony 1)</td><td>0.0201</td><td>9</td><td>0.0585</td><td>76</td><td>0.3224</td><td>339</td><td>0.4975</td><td>472</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Test Device 4: J23101+I13504 (Colony 2)</td><td>0.0257</td><td>16</td><td>0.0628</td><td>84</td><td>0.3267</td><td>340</td><td>0.4900</td><td>471</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Test Device 5: J23106+I13504 (Colony 1)</td><td>0.0263</td><td>7</td><td>0.0804</td><td>46</td><td>0.4000</td><td>106</td><td>0.5413</td><td>120</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Test Device 5: J23106+I13504 (Colony 2)</td><td>0.0269</td><td>6</td><td>0.0850</td><td>45</td><td>0.3789</td><td>94</td><td>0.5285</td><td>111</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Test Device 6: J23117+I13504 (Colony 1)</td><td>0.0265</td><td>1</td><td>0.0959</td><td>8</td><td>0.3967</td><td>15</td><td>0.4837</td><td>2</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Test Device 6: J23117+I13504 (Colony 2)</td><td>0.0243</td><td>-1</td><td>0.0883</td><td>5</td><td>0.4016</td><td>13</td><td>0.5212</td><td>10</td>
 +
                        </tr>
 +
                        </tbody>
 +
                    </table>
 +
                    <p style="color: gray;text-align: center;">Table 2. Raw data of Abs600 and Fluorescence measurement</p>
 +
                    <br/>
 +
                    <table class="table">
 +
                    <thead>
 +
                    <tr>
 +
                    <th>&nbsp;</th><th colspan="2" style="text-align: center;">0h</th><th colspan="2" style="text-align: center;">2h</th><th colspan="2" style="text-align: center;">4h</th><th colspan="2" style="text-align: center;">6h</th>
 +
                    </tr>
 +
                    </thead>
 +
                    <tbody>
 +
                    <tr>
 +
                    <th>&nbsp;</th><th>average</th><th>SD</th><th>average</th><th>SD</th><th>average</th><th>SD</th><th>average</th><th>SD</th>
 +
                    </tr>
 +
                    <tr>
 +
                        <td>Negative Control (Colony 1)</td><td>232</td><td>60</td><td>75</td><td>28</td><td>27</td><td>6</td><td>23</td><td>6</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Negative Control (Colony 2)</td><td>114</td><td>11</td><td>59</td><td>25</td><td>34</td><td>4</td><td>22</td><td>3</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Positive Control (Colony 1)</td><td>920</td><td>180</td><td>827</td><td>222</td><td>1013</td><td>75</td><td>994</td><td>87</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Positive Control (Colony 2)</td><td>720</td><td>40</td><td>911</td><td>86</td><td>867</td><td>81</td><td>960</td><td>15</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Test Device 1: J23101.BCD2.E0040.B0015 (Colony 1)</td><td>1636</td><td>324</td><td>1748</td><td>295</td><td>1394</td><td>80</td><td>1388</td><td>108</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Test Device 1: J23101.BCD2.E0040.B0015 (Colony 2)</td><td>1462</td><td>64</td><td>1227</td><td>80</td><td>1248</td><td>64</td><td>1239</td><td>28</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Test Device 2: J23106.BCD2.E0040.B0015 (Colony 1)</td><td>1190</td><td>212</td><td>1489</td><td>231</td><td>1413</td><td>69</td><td>1763</td><td>92</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Test Device 2: J23106.BCD2.E0040.B0015 (Colony 2)</td><td>716</td><td>103</td><td>1469</td><td>163</td><td>1250</td><td>54</td><td>1621</td><td>83</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Test Device 3: J23117.BCD2.E0040.B0015 (Colony 1)</td><td>27</td><td>61</td><td>112</td><td>50</td><td>49</td><td>7</td><td>49</td><td>11</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Test Device 3: J23117.BCD2.E0040.B0015 (Colony 2)</td><td>282</td><td>108</td><td>148</td><td>45</td><td>52</td><td>3</td><td>45</td><td>11</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Test Device 4: J23101+I13504 (Colony 1)</td><td>461</td><td>76</td><td>1319</td><td>170</td><td>1052</td><td>27</td><td>947</td><td>86</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Test Device 4: J23101+I13504 (Colony 2)</td><td>617</td><td>136</td><td>1350</td><td>189</td><td>1042</td><td>73</td><td>962</td><td>51</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Test Device 5: J23106+I13504 (Colony 1)</td><td>280</td><td>175</td><td>576</td><td>77</td><td>266</td><td>16</td><td>221</td><td>14</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Test Device 5: J23106+I13504 (Colony 2)</td><td>215</td><td>93</td><td>530</td><td>41</td><td>249</td><td>20</td><td>211</td><td>12</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Test Device 6: J23117+I13504 (Colony 1)</td><td>34</td><td>73</td><td>78</td><td>41</td><td>38</td><td>8</td><td>4</td><td>8</td>
 +
                        </tr>
 +
                        <tr>
 +
                        <td>Test Device 6: J23117+I13504 (Colony 2)</td><td>-55</td><td>102</td><td>60</td><td>23</td><td>33</td><td>3</td><td>20</td><td>4</td>
 +
                        </tr>
 +
                    </tbody>
 +
                    </table>
 +
                    <p style="color: gray;text-align: center;">Table 3. Data of Fluorescence/Abs600</p>
 +
                    <p style="color: gray;text-align: center;">
 +
                        <img src="https://static.igem.org/mediawiki/2017/4/4d/T--OUC-China--FI_Abs600_for_8_devices.FI_Abs%EF%BC%88AU%EF%BC%89.png" class="img-thumbnail"><br/>
 +
                        Figure 2.Average level of devices
 +
                    </p>
  
−
             <blockquote class="ouc-heading"><h3 id="results">Results</h3></blockquote>
+
             <h2 id="discussion" class="ouc-heading"><strong>Discussion</strong></h2>
 +
            <p style="font-size:20px;">Device 2 and device 3 have strong fluorescence. Device 4 and device 5 have moderate fluorescence intensity. The device 3 and the device 6 are weakly fluorescent.</p>
 +
       
  
  
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Latest revision as of 13:58, 18 November 2017

InterLab

Team
Project
Proof of concept
Basic fermentation Adhesion platform MINI-GRE
Model
Parts
Safety
Human Practice

Interlab

Background

Fluorescence data can not be directly compared, it is difficult to repeat the measurement. Different instruments, different settings and different measurement environments make this difficult. Through the Interlab study, we expect to have uniform fluorescence measurement steps and standards. These protocols solve this problem by establishing absolute units on the plate reader.

Design

OD 600 Reference point
Using LUDOX-S40 as a reference, the ratiometric conversion factor of Abs and OD600 can be obtained.
Protocol fluorescein fluorescence standard curve
By means of the gradient of the fluorescence standard curve, a method of obtaining a fluorescence correction of the cell can be obtained.
Cell measurement
At four time points, the fluorescence intensity of the eight circuits was measured.

Materials and Method

Plasmids used
Kit Plate 6 InterLab Part Locations
Positive Control (BBa_I20270): well 20B
Negative Control (BBa_R0040): well 20D
Test Device 1(BBa_J364000): well 20F
Test Device 2(BBa_J364001): well 20H
Test Device 3(BBa_J364002): well 20J
Test Device 4(BBa_J364003): well 20L
Test Device 5(BBa_J364004): well 20N
Test Device 6(BBa_J364005): well 20P
Strain
E.coli Competent DH5α （ TaKaRa Code : D9057）
Materials
1ml LUDOX (provided in kit) H20
Fluorescein (provided in kit) 10ml 1xPBS96 well plate, black with flat bottom
LB (Luria Bertani) media Chloramphenicol
50 ml Falcon tube Incubator at 37°C 1.5 ml eppendorf tubes for sample storage Ice bucket with ice Pipettes
Machines
Tecan Spark™ 10M
Software
Microsoft Office EXCEL

Protocol

InterLab_2017_Plate_Reader_Protocol

Results

OD 600 Reference point

 LUDOX-HS40H2O
Replicate 10.04970.0422
Replicate 20.05180.0495
Replicate 30.05460.0428
Replicate 40.05210.0569
Arith. Mean0.052050.04785
Corrected Abs6000.0042 
Reference OD6000.0425 
OD600/Abs60010.1190476 

Table 1. OD600 Reference Point


Protocol fluorescein fluorescence standard curve



Figure 1.Fluorescein fluorescence standard curve


Cell measurement


 0h2h4h6h
 Abs600FIAbs601FIAbs602FIAbs603FI
Negative Control (Colony 1)0.030170.115680.5106140.745317
Negative Control (Colony 2)0.040850.135280.5184170.732616
Positive Control (Colony 1)0.0308280.0805650.47154760.7293722
Positive Control (Colony 2)0.0437310.14981360.49564280.7049677
Test Device 1: J23101.BCD2.E0040.B0015 (Colony 1)0.0283460.08831520.43586060.6846948
Test Device 1: J23101.BCD2.E0040.B0015 (Colony 2)0.0261380.09691190.37194640.5960738
Test Device 2: J23106.BCD2.E0040.B0015 (Colony 1)0.0378440.11201640.50907180.71451258
Test Device 2: J23106.BCD2.E0040.B0015 (Colony 2)0.0223160.07881150.40455060.5945963
Test Device 3: J23117.BCD2.E0040.B0015 (Colony 1)0.032310.0945100.4482220.570428
Test Device 3: J23117.BCD2.E0040.B0015 (Colony 2)0.015640.053780.3399180.525424
Test Device 4: J23101+I13504 (Colony 1)0.020190.0585760.32243390.4975472
Test Device 4: J23101+I13504 (Colony 2)0.0257160.0628840.32673400.4900471
Test Device 5: J23106+I13504 (Colony 1)0.026370.0804460.40001060.5413120
Test Device 5: J23106+I13504 (Colony 2)0.026960.0850450.3789940.5285111
Test Device 6: J23117+I13504 (Colony 1)0.026510.095980.3967150.48372
Test Device 6: J23117+I13504 (Colony 2)0.0243-10.088350.4016130.521210

Table 2. Raw data of Abs600 and Fluorescence measurement


 0h2h4h6h
 averageSDaverageSDaverageSDaverageSD
Negative Control (Colony 1)232607528276236
Negative Control (Colony 2)114115925344223
Positive Control (Colony 1)92018082722210137599487
Positive Control (Colony 2)72040911868678196015
Test Device 1: J23101.BCD2.E0040.B0015 (Colony 1)163632417482951394801388108
Test Device 1: J23101.BCD2.E0040.B0015 (Colony 2)146264122780124864123928
Test Device 2: J23106.BCD2.E0040.B0015 (Colony 1)11902121489231141369176392
Test Device 2: J23106.BCD2.E0040.B0015 (Colony 2)7161031469163125054162183
Test Device 3: J23117.BCD2.E0040.B0015 (Colony 1)2761112504974911
Test Device 3: J23117.BCD2.E0040.B0015 (Colony 2)282108148455234511
Test Device 4: J23101+I13504 (Colony 1)46176131917010522794786
Test Device 4: J23101+I13504 (Colony 2)617136135018910427396251
Test Device 5: J23106+I13504 (Colony 1)280175576772661622114
Test Device 5: J23106+I13504 (Colony 2)21593530412492021112
Test Device 6: J23117+I13504 (Colony 1)3473784138848
Test Device 6: J23117+I13504 (Colony 2)-551026023333204

Table 3. Data of Fluorescence/Abs600


Figure 2.Average level of devices

Discussion

Device 2 and device 3 have strong fluorescence. Device 4 and device 5 have moderate fluorescence intensity. The device 3 and the device 6 are weakly fluorescent.



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