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

 
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<h1 style="margin-bottom: 0px">Date Rape Drugs</h1>
 
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                <h1>Cell-free Sticks - It works on paper</h1>
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                <p>.....</p>
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                <a href="#about" class="btn btn-primary btn-xl page-scroll">Find Out More</a>
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          <a class="navbar-brand" href="#">Project Name</a>
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             <li><a href="#GHBsensor">@fat</a></li>
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            <li><a href="#Blcconsiderations">@mdo</a></li>
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                    <h2 class="section-heading">Project</h2>
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                    <p class="text-faded">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>
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                    <a href="#" class="btn btn-default btn-xl">Get Started!</a>
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<h2> Date rape drug sensor </h2>
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<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>
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<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>
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    <img src="https://static.igem.org/mediawiki/2015/3/38/Bielefeld-CeBiTec_CFPS_EMSA_BlcR.png" alt="EMSA BlcR and BlcR-sfGFP"></a>
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    <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>
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<p> With this proof of functionality, we set out to investigate how the two analytes GBL and GHB can influence the interaction. </p>
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<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>
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<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>
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<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>
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    <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>
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  <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>
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<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>
 
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<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>
 
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    <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>
 
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  <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>
 
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    <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>
 
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  <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>
 
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<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>
 
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<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> 
 
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<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>
 
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<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>
 
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    <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>
 
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  <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>
 
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    <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>
 
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  <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>
 
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                    <h2 class="section-heading">Results</h2>
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                <h2>Overview</h2>
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                <p>...</p>
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                <h2>Final application</h2>
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                <p>It really works on paper...</p>
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                <h2>Modeling</h2>
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                <p>...</p>
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                <h2>Biosafety</h2>
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                <p>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>
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<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>
 
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  <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>
 
 
      
 
      
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                    <h2 class="section-heading">Human Practices</h2>
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    <div id="Blcconsiderations">
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                    <p class="text-faded">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>
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<h2> Considerations </h2>
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                    <a href="#" class="btn btn-default btn-xl">Take a closer look &raquo;</a>
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<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>
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<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>
 
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<h2>Detection of γ-aminobutyrate</h2>
 
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    <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>
 
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        <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>
 
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<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>
 
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<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>
 
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<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>
 
 
      
 
      
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                        <img class="img-circle img-responsive img-center" src="https://static.igem.org/mediawiki/2015/0/0b/Bielefeld-CeBiTec_PT-Teamfoto.jpg" alt="" width="200px" height="200px" style="display: inline">
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                        <h3>Team</h3>
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                        <p class="text-muted">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>
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                        <img class="img-circle img-responsive img-center" src="https://static.igem.org/mediawiki/2015/b/bc/Bielefeld-CeBiTec_notebook_logo.png" alt="" width="120px" height="120px" style="display: inline">
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                        <h3>Notebook</h3>
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                        <p class="text-muted">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>
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                        <h3>Partners</h3>
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                        <p class="text-muted">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>
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    <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>
 
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        <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>
 
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    <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>
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        <figcaption>qRT analysis of the reaction of the <i>gabR</i> and <i>gabT</i> genes in <i>Bacillus subtilis</i> to GBL. </figcaption>
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     <h2> Achievements </h2>
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<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>
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<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>
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<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>
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<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>
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<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>
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<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>
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<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>
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<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>
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<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>
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<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>
 
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Latest revision as of 21:47, 14 September 2015

iGEM Bielefeld 2015


Cell-free Sticks - It works on paper

.....

Find Out More

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.

Get Started!

Results

Overview

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Final application

It really works on paper...

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.

Human Practices

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.

Take a closer look »

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!

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.