Difference between revisions of "Team:Bielefeld-CeBiTec/Sandbox"

Line 3: Line 3:
 
<html>
 
<html>
  
−
<head>
+
<body>
−
<script>
+
−
$("#results").addClass("navbar-active");
+
−
</script>
+
−
</head>
+
−
 
+
−
<body data-spy="scroll" data-target="#pagenav" data-offset="100">
+
  
 
<div class="container">
 
<div class="container">
−
<div class="row">
 
−
<div class="col-md-12">
 
  
−
<div class="jumbotron" style="background-image: url(https://static.igem.org/mediawiki/2015/1/19/Bielefeld-CeBiTec_DateRapeDrugs_Jumbotron2.jpeg)">
+
<div class="jumbotron" style="background-image: url(https://static.igem.org/mediawiki/2015/1/1a/Bielefeld-CeBiTec_jumbotron_startseite.jpeg)">
−
<div class="jumbotron-text">
+
<!-- <h1>Welcome!</h1>    
−
<h1 style="margin-bottom: 0px">Date Rape Drugs</h1>
+
  <p>This wiki is currently under construction.</p>-->
−
<p>...</p>
+
−
</div>
+
 
</div>
 
</div>
  
−
<nav id="pagenav" class="navbar navbar-fixed-bottom"><ul class="nav nav-tabs" role="tablist" style="margin-left: 0px">
+
<div>
−
<li><a href="#GHBsensor">Date rape drug sensor</a></li>
+
<h2 align="center"> Cell-free Sticks - It works on paper. </h2>
  
−
<li><a href="#Blcconsiderations">Considerations</a></li>
+
        <div class="featurette">
−
    <li><a href="#GABA">Detection of γ-aminobutyrate</a></li>
+
            <img class="featurette-image img-responsive pull-left" src="https://static.igem.org/mediawiki/2015/1/18/Bielefeld-CeBiTec_biosensor_overview.jpg" alt="survey result" width="500px" ">
−
</ul>
+
            <p class="lead"><h3>Our project</h3>Our team decided to establish cell-free systems ("cell-free protein synthesis" and "plasmid repressor interaction assay"). For demonstrating the extensibility of our systems we worked with biosensors for heavy metals and we construct a new biosensor for date rape drugs. See what we are doing this year. </p>
−
</nav>
+
        </div>
 +
        <div class="featurette">
 +
            <img class="featurette-image img-responsive pull-right" src="https://static.igem.org/mediawiki/2015/9/93/Bielefeld-CeBiTec_Result_Startseite.jpg" alt="survey result" width="300px" ">
 +
            <p class="lead"><h3>Our results</h3>Our aim is to establish a universally usable and cell-free biosensor on paper. We could demonstrate that some of our biosensors can be detected on paper. Take a look how we achieved our goal successfully. </p>
 +
        </div>
 +
        <div class="featurette">
 +
            <img class="featurette-image img-responsive pull-left" src="https://static.igem.org/mediawiki/2015/0/0b/Bielefeld-CeBiTec_PT-Teamfoto.jpg" alt="survey result" width="500px" ">
 +
            <p class="lead"><h3>Our team</h3>We are ten master students studying molecular biotechnology, genome-based system biology and molecular cell biology. Curious who we are? Get to know us better!</p>
 +
        </div>
 +
        <div class="featurette">
 +
            <img class="featurette-image img-responsive pull-right" src="https://static.igem.org/mediawiki/2015/b/b2/Bielefeld-Cebitec_Dual-use-logo-schmal.png" alt="survey result" width="500px">
 +
            <p class="lead"><h3>Human practices projects</h3>A project is not only lab work. As scientists we need to take responsiblity of what we published and how we appear in public. Among other things we did a dual use essay as we want iGEM to be more aware of biosecurity. Read how iGEM can be a role model for dual use issues. </p>
 +
        </div>
 +
<div class="featurette">
 +
            <img class="featurette-image img-responsive pull-left" src="https://static.igem.org/mediawiki/2015/4/46/Bielefeld-CeBiTec_Modeling_Biosensor_large.png" alt="survey result" width="500px" ">
 +
            <p class="lead"><h3>Modeling</h3></p>
 +
        </div>
 +
        <div class="featurette">
 +
            <img class="featurette-image img-responsive pull-right" src="https://static.igem.org/mediawiki/2015/f/f9/Bielefeld-CeBiTec_Biosafety_jumbotron.jpg" width="500px" ">
 +
            <p class="lead"><h3>Biosafety</h3>Our systems should be cell-free. We confirmed through experiments and experts that our systems contained no genetically modified organisms and are safe to use. Let's see what we've done.</p>
 +
</div>
 +
        <div class="featurette">
 +
            <img class="featurette-image img-responsive pull-left" src="data:image/gif;base64,R0lGODlhAQABAIAAAHd3dwAAACH5BAAAAAAALAAAAAABAAEAAAICRAEAOw==" alt="survey result" width="500px" ">
 +
            <p class="lead"><h3>Notebook</h3>We documented everything we have done in the laboratory and lists protocols, organisms, buffers and primers. If you need more information, check this page.</p>
 +
        </div>
 +
        <div class="featurette">
 +
            <img class="featurette-image img-responsive pull-right" src="data:image/gif;base64,R0lGODlhAQABAIAAAHd3dwAAACH5BAAAAAAALAAAAAABAAEAAAICRAEAOw=="survey result" width="500px" ">
 +
            <p class="lead"><h3>Partners</h3>An important aspect of iGEM is collaboration. We collaborated with several iGEM teams and helped them for their project. Moreover, you can see who supports us financially. Take a look!</p>
 +
        </div>
  
−
<div id="GHBsensor">
+
<h2> Achievements </h2>
−
<h2> Date rape drug sensor </h2>
+
<p>
 +
<table style="background-color:transparent; cellspacing=3;">
 +
<tr><td colspan="2">
 +
<tr><td><img src="https://static.igem.org/mediawiki/2015/e/e7/Bielefeld-CeBiTec-Checkbox_red.png" class="check" width="40px"></td><td>Development of a new cell-free assay named <a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/PRIA" target="_blank">Plasmid Repressor Interaction Assay (PRIA)</a><td></tr>
 +
<tr><td><img src="https://static.igem.org/mediawiki/2015/e/e7/Bielefeld-CeBiTec-Checkbox_red.png" class="check" width="40px"></td><td>Establishment of a <a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPSOverview" target="_blank">cell-free protein synthesis (CFPS) system</a> for the iGEM community</td></tr>
 +
<tr><td><img src="https://static.igem.org/mediawiki/2015/e/e7/Bielefeld-CeBiTec-Checkbox_red.png" class="check" width="40px"></td><td>Demonstration of the biosensor system's extensibility through <a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/HeavyMetals">several biosensors</a></td></tr>
 +
<tr><td><img src="https://static.igem.org/mediawiki/2015/e/e7/Bielefeld-CeBiTec-Checkbox_red.png" class="check" width="40px"></td><td>Construction of a new biosensor for the detection of <a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/DateRapeDrugs">date rape drugs</a></td></tr>
 +
<tr><td><img src="https://static.igem.org/mediawiki/2015/e/e7/Bielefeld-CeBiTec-Checkbox_red.png" class="check" width="40px"></td><td>Construction of a new biosensor for <a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/HeavyMetals">heavy metals (Copper) </a>. </td></tr>
 +
<tr><td><img src="https://static.igem.org/mediawiki/2015/e/e7/Bielefeld-CeBiTec-Checkbox_red.png" class="check" width="40px"></td><td>Implementation of a <a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Modeling">model</a> for CFPS.<td></tr>
 +
<tr><td><img src="https://static.igem.org/mediawiki/2015/e/e7/Bielefeld-CeBiTec-Checkbox_red.png" class="check" width="40px"></td><td>Taking up the <a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Practices/DualUse">dual use issue</a> in connection with the topic "date rape drugs".</td></tr>
 +
<tr><td><img src="https://static.igem.org/mediawiki/2015/e/e7/Bielefeld-CeBiTec-Checkbox_red.png" class="check" width="40px"></td><td><a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Collaborations" target="_blank">Help another team</a></td></td></tr>
 +
<tr><td><img src="https://static.igem.org/mediawiki/2015/e/e7/Bielefeld-CeBiTec-Checkbox_red.png" class="check" width="40px"></td><td>We have a <a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Design" target="_blank">functional prototype</a>.</td></td></tr>
 +
</table>
 +
</p>
  
−
<p> We demonstrated that we can detect the date rape drug ingredients &gamma;-butyrolactone (GBL) and &gamma;-hydroxybutyric acid (GHB) with the help of a small protein, BlcR, in combination with its cognate DNA sequence, the blc-operator. We used various methods to characterize the interaction process.  </p>
 
−
<p> We performed <a data-toggle="tooltip" title="Electrophoretic Mobility Shift Assay">EMSA</a> and verified: BlcR binds to the operator site described in <a href="#Pan2013">Pan et al. 2013</a>, even when it is N-terminal fused to sfGFP (see <a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Results/PRIA">PRIA results</a>). </p>
 
  
−
<figure style="margin:auto; width:300px">
 
−
    <img src="https://static.igem.org/mediawiki/2015/3/38/Bielefeld-CeBiTec_CFPS_EMSA_BlcR.png" alt="EMSA BlcR and BlcR-sfGFP"></a>
 
−
    <figcaption> EMSA shifts caused by addition of BlcR protein (see <a href="http://parts.igem.org/Part:BBa_K1758370" target="_blank">BBa_K1758370</a>) and BlcR-sfGFP fusion protein (see <a href="http://parts.igem.org/Part:BBa_K1758204" target="_blank">BBa_K1758204</a>), respectively, to Cy3-labeled blc-operator site. 5 pmol of following proteins were applied: 1: BlcR-sfGFP, 2: ArsR-sfGFP (see <a href="http://parts.igem.org/Part:BBa_K1758203" target="_blank">BBa_K1758203</a>), 3: BlcR, 4: none. </figcaption>
 
−
    </figure>
 
  
  
−
</br>
+
<!-- <h2> Welcome to iGEM 2015! </h2>
−
<p> With this proof of functionality, we set out to investigate how the two analytes GBL and GHB can influence the interaction. </p>
+
<p>Your team has been approved and you are ready to start the iGEM season! </p>
−
 
+
−
<p> GBL and GHB are both toxic to <i>E. coli</i> when their concentration in the medium exceeds a certain limit. We observed that for GHB the tolerable dose is under 1% (v/v), whereas <i>E. coli</i> can live in medium with 3% (v/v) GBL. </p>
+
−
 
+
−
<p> An <i>E. coli</i> strain carrying <a href="http://parts.igem.org/Part:BBa_K1758377" target="_blank">BBa_K1758377</a> in pSB1C3 was induced to express T7 polymerase in medium with different concentrations of either GBL or GHB. As control, medium without GBL nor GHB was used. Induction lead to expression of sfGFP. However, sfGFP coding sequence follows on blc operator sequence. As the strain constitutively expresses BlcR, we expected the fluorescence signal to be higher when GBL or GHB were present in the medium as both analytes interact with BlcR and inhibit its binding to the operator. </p>
+
−
<p> Fluorescence signals of strains that had grown in medium with analytes were slightly higher, except for cultures with 1% GHB which showed inhibited growth. </p>
+
−
 
+
−
<figure style="width: 400px; margin-right: 20px; float: left">
+
−
    <a href="https://static.igem.org/mediawiki/parts/0/0a/Bielelfeld-CeBiTec_Blc-cultivation_in_vivo_sensor_big.png" data-lightbox="in vivo gbl" data-title="Characterization of GBL / GHB sensor in vivo"><img src="https://static.igem.org/mediawiki/parts/f/fe/Bielefeld-CeBiTec_Blc-cultivation_in_vivo_sensor_small.jpg" alt="Characterization of GBL / GHB sensor in vivo"></a>
+
−
  <figcaption> <i>In vivo</i> characterization of GBL / GHB sensor with strain containing <a href="http://parts.igem.org/Part:BBa_K1758377" target="_blank">BBa_K1758377</a>. All experiments were perfomed as triplicates. All samples except "control, not induced" were induced to express T7 polymerase at OD<sub>600</sub> = 0.7-0.8 </figcaption>
+
−
    </figure>
+
−
 
+
−
 
+
−
 
+
−
<div class="row">
+
−
<div class="col-md-12">
+
−
   
+
−
<p> These results indicated that, although a difference could be seen, the device has its limits <i>in vivo</i>. We conducted a <a data-toggle="tooltip" title="Cell free protein synthesis">CFPS</a> with extract from strain constitutivly expressing BlcR. As reporter plasmid, <a href="http://parts.igem.org/Part:BBa_K1758376" target="_blank">BBa_K1758376</a> was used. This plasmid equals our CFPS positive control P<sub>T7</sub>-UTR-sfGFP (see <a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Results/CFPS">CFPS results</a>) except that T7 promoter is followed by the blc-operator. </p>
+
  
 +
<h4>Before you start: </h4>
 +
<p> Please read the following pages:</p>
 +
<ul>
 +
<li>  <a href="https://2015.igem.org/Requirements">Requirements page </a> </li>
 +
<li> <a href="https://2015.igem.org/Wiki_How-To">Wiki Requirements page</a></li>
 +
</ul>
  
−
<p> As well as <i>in vivo</i>, GBL and GHB had detrimental effects on the molecular machinery. 0.3% (v/v) of GBL were sufficient to strongly, but not completely inhibit protein synthesis when we used our standard cell extract. For GHB the effect was even greater, stopping protein synthesis completely at 3% (v/v) final concentration as depicted in the graphs. </p>
+
<div class="highlightBox">
−
</br>
+
<h4> Styling your wiki </h4>
−
 
+
<p>You may style this page as you like or you can simply leave the style as it is. You can easily keep the styling and edit the content of these default wiki pages with your project information and completely fulfill the requirement to document your project.</p>
−
<!-- influence GBL und GHB -->
+
<p>While you may not win Best Wiki with this styling, your team is still eligible for all other awards. This default wiki meets the requirements, it improves navigability and ease of use for visitors, and you should not feel it is necessary to style beyond what has been provided.</p>  
−
<figure style="width: 400px; margin-right: 20px; float: left">
+
−
    <a href="https://static.igem.org/mediawiki/2015/3/32/Bielefeld-CeBiTec_Blc_GBL_influence_big.png" data-lightbox="influence" data-title="Influence of &gamma;-butyrolactone (GBL) on expression of sfGFP in CFPS reaction (t = 60 min)."><img src="https://static.igem.org/mediawiki/2015/4/43/Bielefeld-CeBiTec_Blc_GBL_influence_small.png" alt="bar chart GBL influence"></a>
+
−
  <figcaption>Influence of &gamma;-butyrolactone (GBL) on expression of sfGFP in our standard CFPS reaction (t = 60 min). Positive control: P<sub>T7</sub>-UTR-sfGFP (<a href="http://parts.igem.org/Part:BBa_K1758102" target="_blank">BBa_K1758102</a>). Values are normalized to cell lysate containing sfGFP. </figcaption>
+
−
    </figure>
+
−
   
+
−
<figure style="width: 400px; margin-left: 20px; float: right">
+
−
    <a href="https://static.igem.org/mediawiki/2015/f/ff/Bielefeld-CeBiTec_Blc_GHB_influence_big.png" data-lightbox="influence" data-title="Influence of &gamma;-hydroxybutyrate (GHB) on expression of sfGFP in CFPS reaction (t = 60 min)."><img src="https://static.igem.org/mediawiki/2015/d/dd/Bielefeld-CeBiTec_Blc_GHB_influence_small.png" alt="bar chart GHB influence"></a>
+
−
  <figcaption>Influence of &gamma;-hydroxybutyrate (GHB) on expression of sfGFP in our standard CFPS reaction (t = 60 min). Positive control: P<sub>T7</sub>-UTR-sfGFP (<a href="http://parts.igem.org/Part:BBa_K1758102" target="_blank">BBa_K1758102</a>) </figcaption>
+
−
    </figure>
+
−
   
+
−
</div>
+
 
</div>
 
</div>
  
−
<div class="row">
+
<h4> Editing your wiki </h4>
−
<div class="col-md-12">
+
<p>On this page you can document your project, introduce your team members, document your progress and share your iGEM experience with the rest of the world! </p>
 +
<p> <a href="https://2015.igem.org/wiki/index.php?title=Team:Bielefeld-CeBiTec&action=edit"> Click here to edit this page! </a></p>
 +
<p>See tips on how to edit your wiki on the <a href="https://2015.igem.org/TemplatesforTeams_Code_Documentation">Template Documentation</a> page.</p>  
  
−
<p> This however did not stop us from further testing. We took <i>E. coli</i> that constitutively expressed BlcR (<a href="http://parts.igem.org/Part:BBa_K1758370" target="_blank">BBa_K1758370</a>). In less than a day we cultivated the cells, made cell extract via sonification and performed CFPS with <a href="http://parts.igem.org/Part:BBa_K1758376" target="_blank">BBa_K1758376</a> as reporter. In standard extract, fluorescence signals of our positive control plasmid and BBa_K1758376 were similar. </p>
 
−
<p> The results of the CFPS reaction surpassed all expectations. <i>In vivo</i>, BlcR reacts on GHB and GBL and thereby dissociates from the blc-operator (<a href="#Chai2007">Chai et al. 2007</a>). This effect could be observed when 0.3% GBL was present in the reaction, as the flurescence signal was greater when compared to the reaction without GBL. Still, for higher concentrations of GBL, protein synthesis was inhibited.  </p> 
 
−
<p> GHB also negatively affected protein synthesis in BlcR containing extract. <b>Strikingly however, detrimental effects were far smaller than in standard extract!</b> Especially interesting was that <b>for 3% GHB, the fluorescence signal surpassed the 1% GHB signal</b>. We suppose two reasons that together lead to this effect: When BlcR binds to GHB, on the one hand GHB is removed from the reaction and can not act detrimental on the molecular machinery, and on the other hand the polymerase is no longer blocked by BlcR, that means sfGFP can be expressed. </p>
 
−
<p> When we normalized the signals from BlcR containing extract on our standard extract in which GHB was strongly inhibiting, the effect of BlcR could not be overlooked. Consistent with findings from <a href="#Chai2007">Chai et al. 2007</a>, BlcR reaction on GHB is stronger than on GBL.  </p>
 
  
−
<figure style="width: 400px; margin-right: 20px; float: left">
+
<h4>Templates </h4>
−
    <a href="https://static.igem.org/mediawiki/2015/3/3d/Bielefeld-CeBiTec_Blc_GHB_effect_blcR_extract_big.png" data-lightbox="GHBsensor" data-title="Influence of &gamma;-hydroxybutyrate (GHB) on expression of sfGFP in extract containing BlcR (t = 60 min)"><img src="https://static.igem.org/mediawiki/2015/6/6f/Bielefeld-CeBiTec_Blc_GHB_effect_blcR_extract_small.png" alt="GHB in BlcR extract"></a>
+
<p> This year we have created templates for teams to use freely. More information on how to use and edit the templates can be found on the
−
  <figcaption> Influence of &gamma;-hydroxybutyrate (GHB) on expression of sfGFP in extract containing BlcR (t = 60 min). DNA template was <a href="http://parts.igem.org/Part:BBa_K1758376" target="_blank">BBa_K1758376</a>.   </figcaption>
+
<a href="https://2015.igem.org/TemplatesforTeams_Code_Documentation">Template Documentation </a> page.</p>  
−
    </figure>
+
  
  
−
<figure style="width: 400px; margin-left: 20px; float: right">
+
<h4>Tips</h4>
−
    <a href="https://static.igem.org/mediawiki/2015/9/9b/Bielefeld-CeBiTec_Blc_GHB_sensor_big.png" data-lightbox="GHBsensor" data-title="Extract containing BlcR reveals response to GHB when the observed fluorescence signal is normalized to signal generated in our standard extract (t = 60 min)"><img src="https://static.igem.org/mediawiki/2015/4/49/Bielefeld-CeBiTec_Blc_GHB_sensor_small.png" alt="GHB induces fluorescence"></a>
+
<p>This wiki will be your team’s first interaction with the rest of the world, so here are a few tips to help you get started: </p>
−
  <figcaption> Extract containing BlcR reveals response to GHB when the observed fluorescence signal is normalized to signal generated in our standard extract (t = 60 min). </figcaption>
+
<ul>
−
    </figure>
+
<li>State your accomplishments! Tell people what you have achieved from the start. </li>
−
   
+
<li>Be clear about what you are doing and how you plan to do this.</li>
−
    </div>
+
<li>You have a global audience! Consider the different backgrounds that your users come from.</li>
−
</div>
+
<li>Make sure information is easy to find; nothing should be more than 3 clicks away.  </li>
 +
<li>Avoid using very small fonts and low contrast colors; information should be easy to read. </li>
 +
<li>Start documenting your project as early as possible; don’t leave anything to the last minute before the Wiki Freeze. For a complete list of deadlines visit the <a href="https://2015.igem.org/Calendar_of_Events">iGEM 2015 calendar</a> </li>
 +
<li>Have lots of fun! </li>
 +
</ul>  
  
−
<div class="row">
 
−
<div class="col-md-12">
 
−
   
 
−
<p> We therefore demonstrated that we can detect GHB at concentrations of 1% and 3% by normalizing the fluorescence signal to a control reaction. As our CFPS system is very <a href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Results/CFPS#robustness">robust</a> even at ethanol concentrations of 5%, we can say that we built a cell-free sensor for GHB that can be used to detect the noxious substance in liquids. </p>
 
−
  <p> In the final application, the potential of our sensor became evident. In our paper-based CFPS reaction, water that contained 1% (v/v) GHB was used for rehydration. Fluorescence signals were measured, data quickly evaluated and our app demonstrated that the water was contaminated with date rape drugs ingredients. For detail see <a href="">our final sensor approach</a> </p>
 
−
   
 
−
    </div>
 
−
</div>
 
  
−
</div> <!-- end of GHB sensor div -->
+
<h4>Inspiration</h4>
 +
<p> You can also view other team wikis for inspiration! Here are some examples:</p>
 +
<ul>
 +
<li> <a href="https://2014.igem.org/Team:SDU-Denmark/"> 2014 SDU Denmark </a> </li>
 +
<li> <a href="https://2014.igem.org/Team:Aalto-Helsinki">2014 Aalto-Helsinki</a> </li>
 +
<li> <a href="https://2014.igem.org/Team:LMU-Munich">2014 LMU-Munich</a> </li>
 +
<li> <a href="https://2014.igem.org/Team:Michigan"> 2014 Michigan</a></li>
 +
<li> <a href="https://2014.igem.org/Team:ITESM-Guadalajara">2014 ITESM-Guadalajara </a></li>
 +
<li> <a href="https://2014.igem.org/Team:SCU-China"> 2014 SCU-China </a></li>
 +
</ul>
  
−
    <div id="Blcconsiderations">
+
<h4> Uploading pictures and files </h4>
−
<h2> Considerations </h2>
+
<p> You can upload your pictures and files to the iGEM 2015 server. Remember to keep all your pictures and files within your team's namespace or at least include your team's name in the file name. <br />
−
<p> Our standard cell extract was sucessfully optimized in the course of our project. A future aim for our GHB sensor would be the optimization of BlcR containing <i>E. coli</i> cell extract as well as tests with purified BlcR. A higher fluorescence output would make detection with our measurement prototype and app easier.  </p>
+
When you upload, set the "Destination Filename" to <code>Team:YourOfficialTeamName/NameOfFile.jpg</code>. (If you don't do this, someone else might upload a different file with the same "Destination Filename", and your file would be erased!)</p>
−
   
+
−
<p> Furthermore, the results presented here clearly show that a CFPS based <i>in vitro</i> approach has many advantages over an <i>in vivo</i> approach: Time saving experiments, analysis of toxic substances and easily tunable reactions in a minute scale comprise enormous chances and potential in various research areas.  </p>
+
  
−
</div>
+
<a href="https://2015.igem.org/Special:Upload">CLICK HERE TO UPLOAD FILES</a>
  
−
</div>
 
−
</div>
 
  
  
−
<div id="GABA">
+
</div></div>These are the closing tags for div id="mainContainer" and div id="contentContainer". The corresponding opening tags appear in the template that is {{included}} at the top of this page.
−
<div class="row">
+
−
<div class="col-md-12">
+
−
<h2>Detection of γ-aminobutyrate</h2>
+
  
−
    <figure style="width: 600px; margin-left: 20px; float: right">
+
</div>-->
−
    <a href="https://static.igem.org/mediawiki/2015/d/dd/Bielefeld-CeBiTec_GABA_induction_large.png" data-lightbox="daterapedrugs" data-title="Induction of <i>B. subtilis</i> GABA sensor with GABA. 5 mL cultures in M9 with different GABA concentrations were grown overnight and RFP fluorescence was measured in a plate reader. The error bars represent the standard deviation of three biological replicates."><img src="https://static.igem.org/mediawiki/2015/3/3c/Bielefeld-CeBiTec_GABA_induction_small.png" alt="Induction of GABA sensor"></a>
+
−
        <figcaption>Induction of B. subtilis GABA sensor with GABA. 5 mL cultures in M9 with different GABA concentrations were grown overnight and RFP fluorescence was measured in a plate reader. The error bars represent the standard deviation of three biological replicates.</figcaption>
+
−
    </figure>
+
−
<p>As an alternative to the direct detection of GBL or GHB, we constructed biosensors for the structural analogue γ-aminobutyrate (GABA). By enzymatically converting GBL to GABA, it would be possible to use such a sensor for the detection of date rape drugs as well.</p>
+
−
<p>We found out that <i>Bacillus subtilis</i> and <i>Rhizobium leguminosarum</i> posses operons that can be induced by GABA. We obtained the genes for the responsible proteins and the inducable promoters by gene synthesis and placed mRFP1 under the control of the inducable promoters. After growing overnight cultures with 10 mg/L GABA, we observed that the cell pellets of the <i>B. subtilis</i> sensor were clearly red, while the pellet of the <i>R. leguminosarum</i> sensor did not differ from the negative control. Consequently, we decided to work with the <i>B. subtilis</i> sensor. Upon further characterization of the biosensor, we noticed that the background signal in LB medium was very high, possibly because it containes traces of GABA. The background signal in M9 medium was considerably lower and we observed a clear induction by GABA when growing overnight cultures with different GABA concentrations and measuring the RFP fluorescence in a plate reader. A reaction was observable down to concentrations of 1 mg/L.</p>
+
  
−
</div>
 
−
</div>
 
−
 
−
<div class="row">
 
−
<div class="col-md-12">
 
−
<p>We also tested whether it is possible to induce the biosensor with GBL. However, we observed no signal with three different GBL concentrations. In contrast, a qRT PCR showed an upregulation of the <i>gabT</i> gene in <i>B. subtilis</i>. This gene is usually activated by the transcription factor GabR in the presence of GABA. Our biosensor is based on <i>gabR</i> and the <i>gabT</i> promoter, so we had expected a similar response of the operon and the biosensor. As <i>B. subtilis</i> is able to metabolize GBL, we assume that this metabolism resulted in an induction of the <i>gabTD</i> operon. With regard to our biosensor, this confirms that the GABA sensor can be used to detect date rape drugs in combination with an enzymatic conversion.</p>
 
−
   
 
−
</div>
 
−
</div>
 
−
 
−
<div class="row">
 
−
<div class="col-md-6">
 
−
   
 
−
    <figure style="width: 500px">
 
−
    <a href="https://static.igem.org/mediawiki/2015/9/9f/Bielefeld-CeBiTec_GABA_GBLeffect_large.png" data-lightbox="daterapedrugs" data-title="Effect of GBL on <i>B. subtilis</i> GABA sensor. 5 mL cultures in M9 with different GBL concentrations were grown overnight and RFP fluorescence was measured in a plate reader. The error bars represent the standard deviation of three biological replicates."><img src="https://static.igem.org/mediawiki/2015/8/89/Bielefeld-CeBiTec_GABA_GBLeffect_small.png" alt="Effect of GBL on GABA sensor"></a>
 
−
        <figcaption>Effect of GBL on B. subtilis GABA sensor. 5 mL cultures in M9 with different GBL concentrations were grown overnight and RFP fluorescence was measured in a plate reader. The error bars represent the standard deviation of three biological replicates.</figcaption>
 
−
    </figure>
 
−
   
 
−
</div>
 
−
<div class="col-md-6">
 
−
   
 
−
    <figure style="width: 500px">
 
−
    <a href="https://static.igem.org/mediawiki/2015/5/5f/Bielefeld-CeBiTec_GABA_qRT_large.png" data-lightbox="daterapedrugs" data-title=""><img src="https://static.igem.org/mediawiki/2015/4/4b/Bielefeld-CeBiTec_GABA_qRT_small.png" alt="Effect of GBL on GABA sensor"></a>
 
−
        <figcaption>qRT analysis of the reaction of the <i>gabR</i> and <i>gabT</i> genes in <i>Bacillus subtilis</i> to GBL. </figcaption>
 
−
    </figure>
 
−
   
 
−
</div>
 
−
</div>
 
−
   
 
−
</div>
 
−
 
−
<div class="row footer">
 
−
<div class="col-md-2 col-md-offset-10 text-center">
 
−
<a type="button" class="btn btn-default btn-next" href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Modeling"><img src="https://static.igem.org/mediawiki/2015/5/51/Bielefeld-CeBiTec_iGEM_logo.png"><p>No iGEM project without ...</p></a>
 
−
</div>
 
−
</div>
 
−
</div>
 
 
</body>
 
</body>
  

Revision as of 18:26, 14 September 2015

iGEM Bielefeld 2015


Cell-free Sticks - It works on paper.

survey result

Our project

Our team decided to establish cell-free systems ("cell-free protein synthesis" and "plasmid repressor interaction assay"). For demonstrating the extensibility of our systems we worked with biosensors for heavy metals and we construct a new biosensor for date rape drugs. See what we are doing this year.

survey result

Our results

Our aim is to establish a universally usable and cell-free biosensor on paper. We could demonstrate that some of our biosensors can be detected on paper. Take a look how we achieved our goal successfully.

survey result

Our team

We are ten master students studying molecular biotechnology, genome-based system biology and molecular cell biology. Curious who we are? Get to know us better!

survey result

Human practices projects

A project is not only lab work. As scientists we need to take responsiblity of what we published and how we appear in public. Among other things we did a dual use essay as we want iGEM to be more aware of biosecurity. Read how iGEM can be a role model for dual use issues.

survey result

Modeling

Biosafety

Our systems should be cell-free. We confirmed through experiments and experts that our systems contained no genetically modified organisms and are safe to use. Let's see what we've done.

survey result

Notebook

We documented everything we have done in the laboratory and lists protocols, organisms, buffers and primers. If you need more information, check this page.

Partners

An important aspect of iGEM is collaboration. We collaborated with several iGEM teams and helped them for their project. Moreover, you can see who supports us financially. Take a look!

Achievements

Development of a new cell-free assay named Plasmid Repressor Interaction Assay (PRIA)
Establishment of a cell-free protein synthesis (CFPS) system for the iGEM community
Demonstration of the biosensor system's extensibility through several biosensors
Construction of a new biosensor for the detection of date rape drugs
Construction of a new biosensor for heavy metals (Copper) .
Implementation of a model for CFPS.
Taking up the dual use issue in connection with the topic "date rape drugs".
Help another team
We have a functional prototype.