Team:Paris Bettencourt/Designs

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<h1>Designs overview</h1>
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<h2>Introduction</h2>
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<p>This page is a summing up of the design we have builded and the explanation of the approaches used to build these designs.</p>
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<table>
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<tr>
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<td>
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<p><b>Our goal for this summer is to characterize the travel of various molecules through nanotubes. In order to do so, we developed several designs. In this page, we explain the principle of the different designs we have built.</b></p>
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<h1>Designs for a direct observation</h1>
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<p>In the original paper <a href="https://2011.igem.org/Team:Paris_Bettencourt/Designs#references">[1]</a>, the authors directly observed  the passage of molecules through the nanotubes. The idea behind our approach is to use <em>synthetic biology methods</em> to improve the sensitivity and the resolution of these experiments. Relying on <em>signal amplification</em> and natural, as well as artificial, <em>bistable switches</em>, we want to detect, with a resolution at the molecular level, the passage from <b>one emitter cell</b> to <b>the receiver</b>.</p>
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<a href="https://2011.igem.org/File:Nanotube_and_GFP.jpeg"><img src="../wiki/images/e/ed/Nanotube_and_GFP.jpeg" style="float:right; width: 250px; margin: 15px;"></a>
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</td>
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    <td style="width:200px;"><center><img src="https://static.igem.org/mediawiki/2011/8/86/Logo_projet.png" alt="our logo" width="150px"></center></td>
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</tr>
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</table>
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<h2>Main questions</h2>
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<p>The principle of these designs is to observe directly the molecule that passes from a producer cell to a receptor cell through the nanotubes. There is no signal amplification in this step.</p>
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<p>The nanotube discovery is very recent. Lots of questions remain unsolved concerning the mechanism behind the formation of the tubes, the <em>efficiency of the transfer</em>, the extent of the process. The existence of the tubes themselves remains a subject of controversy within the scientific community.</p>
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<p>We currently have 2 designs for it:</p>
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<p>We aim to bring additional proof of their existence and better characterize the extent of this phenomenon. Here is the list of the questions we have been asking ourselves, and that our designs aim to answer:<p>
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<table>
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<tr>
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<td style="width:200px; text-align:center"><img style="width:150px" src="https://static.igem.org/mediawiki/2011/2/2b/Question_mark_button.png">
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  </td>
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<td>
<ul>
<ul>
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<li><a href="Team:Paris_Bettencourt/Antibiotic_diffusion">The Antibiotics resistance experiences</a> The principle is to put two strains of bacteria that present different antibiotic resistances in the same biofilm. After a growth time together, they are exposed to the two antibiotics corresponding to the resistances. They can resist together through a cooperative effect involving the exchange of antibiotic resistance enzymes through the nanotubes.</li>
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<li>What is the <em>nature</em> of the process? (passive or active transport)</li>
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<li>[[Team:Paris_Bettencourt/GFPLac_diffusion|The GFP-LacI fusion:]] The principle is to diffuse a GFP-LacI fusion protein from one cell that produces it to a neighboring cell that is without color and contains a plasmid with numerous LacO operons. The GFP that enters the neighboring cell via the nanotubes will then be concentrate on the plasmid giving a more intense fluorescence.</li>
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<li>What kinds of molecules can pass through the nanotubes?</li>
 +
<li>Does the communication happen only between <i>B. subtilis</i>? Or can it also exist between different species?</li>
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<li>If a communication can be established with Gram negative bacteria, is the communication happening through the periplasm or the cytoplasm?</li>
</ul>
</ul>
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<br>
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</td>
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</tr>
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</table>
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<p>There is also the question of the formation of the tubes. However, this specific problem would require a lot more than a summer and a team of undergraduates to be fully investigated. We therefore choose not to focus too much on it, even though we have some <a href="https://2011.igem.org/Team:Paris_Bettencourt/Modeling/Assisted_diffusion">ideas</a>.
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<h1>Designs for nanotube characterization</h1>
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<h2>Specification of our designs</h2>
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<p>The aim of this state is to characterize the nanotube communication. The idea is to pass molecules of different sizes and to measure the interval of time between the apparition of the two monitors which is linked to the diffusion time through the nanotube.</p>
 
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<a href="https://2011.igem.org/File:Step1_principle.jpg"><img src="../wiki/images/1/1c/Step1_principle.jpg" style="width: 600px; margin-left: 185px;"></a></br>
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<p>We started designing the project with these previously stated questions in mind. Using several molecules of <em>different sizes and nature</em>, from a T7 RNA polymerase to ComS, we aim to test the <em>nature, size and number of the molecules we want to diffuse through the nanotubes</em>.</p>
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[[File:Step1_principle.jpg|thumb|center|upright=3.0|General plan for the experiment design to characterize the nanotubes]]
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<p>First, we put the two constructions in the same cell and induce their expression with different quantities of IPTG. Then we measure the time between the apparition of monitor 1 (RFP) and monitor 2 (GFP). We repeat the experiment, but with the two constructs separately introduced into two different cells. There should be an increase in the delay of apparition of the second monitor due to the necessary diffusion time through the nanotube.</p>
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<p>We decided that our constructs should follow the global idea summed up in the following scheme:</p>
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<p>We expect the time to increase with the size of the molecule as the diffusion coefficient is D = K/R where K is a constant and R the Stoke radius.</p>
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<center><a href="https://2011.igem.org/File:Step1_principle.jpg"><img src="https://static.igem.org/mediawiki/2011/e/e6/Schema-presentation.png"></a></center></br>
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<p><center><b>General plan for the experiment design to characterize the nanotubes</center></b></p>
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<p>We try to demonstrate and characterize the communication through the nanotube system for Bacillus Subtilis, but also between B. Subtilis and E. Coli. The designs are classified by type of demonstration.</p>
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<p>As explained in our <a href="https://2011.igem.org/Team:Paris_Bettencourt/Modeling">modeling section</a>, diffusion seems to be too quick comparatively to genetic networks response time to be accurately measured. We therefore focused on testing the <em>nature</em>, the <em>number</em> and the <em>size</em> of the molecules we try to pass trough the nanotubes.</p>
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<br>
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<p>In <i>B.subtilis</i>, nanotubes are reported allow transfer to the cytoplasm. We had to find molecules that can be expressed in the first cell and then trigger a genetic circuit in the second cell. The image below illustrates the idea:</p>
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<h2>Design for Subtilis-Subtilis communication</h2>
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<a href="https://2011.igem.org/File:cytoplasm_cytoplasm_communication.jpg"><img src="../wiki/images/4/49/Cytoplasm_cytoplasm_communication.jpg" style="width: 400px; margin-left: 235px;"></a></br>
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<p><center><b>Schematic of the supposed connection between the cells via the nanotubes</b></center></p>
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<p>In B. Subtilis, the nanotubes directly connect the cytoplasms of the two cells. Thus, simple designs with cytoplasmic proteins can be used.</p>
 
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<a href="https://2011.igem.org/File:cytoplasm_cytoplasm_communication.jpg"><img src="../wiki/images/4/49/Cytoplasm_cytoplasm_communication.jpg" style="width: 600px; margin-left: 185px;"></a></br>
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<h2>Genetic devices we tested</h2>
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[[File:cytoplasm_cytoplasm_communication.jpg|thumb|center|upright=2.0|Schematic of the supposed connection between the cells via the nanotubes]]
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-
<p>The designs we have made so far are the following, classified by the size of the molecule we want to pass through the tubes:</p>
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<p>Using these specifications, we designed several sensitive genetic circuits that can be trigerred by molecules of various sizes. The molecules chosen cover 1 orders of magnitude of radius.</p>
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 +
<br />
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<br />
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<center>
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<img src="https://static.igem.org/mediawiki/2011/d/d7/Size_chart.png" style="width: 900px;">
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</center>
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<br />
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<br />
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<table>
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<tr>
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<th style="width:200px;"></th>
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<th style="text-align:center;"><b>Description</b></th>
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<th style="width:100px;text-align:center;"><b>Type of switch</b></th>
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<th style="width:100px;text-align:center;"><b>B. subilis/<br/>E. coli<br/>compatibility</b></th>
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</tr>
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<tr>
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  <td style="text-align:center;"><a href="https://2011.igem.org/Team:Paris_Bettencourt/T7_diffusion"><img style="width:150px; margin-top:20px;" src="https://static.igem.org/mediawiki/2011/e/e4/T7_button.png"></a>
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  </td>
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  <td><em><a href="https://2011.igem.org/Team:Paris_Bettencourt/T7_diffusion">T7 polymerase diffusion</a></em> The T7 RNA polymerase is the RNA polymerase of the T7 phage. This is a big molecule, that recognizes a very specific promoter orthogonal to <i>B.subtilis</i>.
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  </td>
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<td style="text-align:center;">Irreversible<br/>amplification</td>
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<td style="text-align:center;">Yes</td>
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</tr>
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<tr>
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  <td style="text-align:center"><a href="https://2011.igem.org/Team:Paris_Bettencourt/tRNA_diffusion"><img style="width:150px; margin-top:20px;" src="https://static.igem.org/mediawiki/2011/5/53/TRNAamber-button.png"></a>
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  </td>
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  <td><em><a href="https://2011.igem.org/Team:Paris_Bettencourt/tRNA_diffusion">Amber suppressor tRNA diffusion</a></em> The principle of this design is to produce in one cell an amber supressor tRNA that will diffuse through the nanotubes. The receptor cell holds the gene for T7 with amber stop codons that cannot be translated into a functional protein as long as the tRNA amber suppressor is not present in the cell. Once expressed, the functional amber T7 RNA polymerase will trigger the T7 amplification system.
 +
  </td>
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<td style="text-align:center;">Irreversible<br/>amplification</td>
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<td style="text-align:center;">Yes</td>
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</tr>
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<tr>
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  <td style="text-align:center"><a href="https://2011.igem.org/Team:Paris_Bettencourt/SinOp"><img style="width:150px; margin-top:20px;" src="https://static.igem.org/mediawiki/2011/a/aa/SinOp-button.png"></a>
 +
  </td>
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  <td><em><a href="https://2011.igem.org/Team:Paris_Bettencourt/SinOp">KinA diffusion</em></a> The Sin operon system controls the sporulation switch of <i>B. subtilis</i>. Making it diffuse from a non sporulating strain to a receiver cell makes the receiver cell to sporulate. We also use fluorescent sporulation reporters to monitor the experiment properly.
 +
  </td>
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<td style="text-align:center;">Switch</td>
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<td style="text-align:center;">Mono-<br/>directional</td>
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</tr>
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<tr>
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  <td style="text-align:center"><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/Lambda_switch"><img style="width:150px; margin-top:20px;" src="https://static.igem.org/mediawiki/2011/9/94/Lambda_switch-button.png"></a>
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  </td>
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  <td><em><a href="https://2011.igem.org/Team:Paris_Bettencourt/Lambda_switch">Lambda switch design</em></a> The CI is a regulation protein from the lambda phage switch. Make it diffuse would change the state of the toogle switch from the push-on push-off system, from the <a href="https://2007.igem.org/Peking">PKU 2007</a>.
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  </td>
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<td style="text-align:center;">Switch</td>
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<td style="text-align:center;">Mono-<br/>directional</td>
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</tr>
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<tr>
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  <td style="width:200px; text-align:center"><a href="https://2011.igem.org/Team:Paris_Bettencourt/GFPLac_diffusion"><img style="width:150px" src="https://static.igem.org/mediawiki/2011/d/d0/YFP_concentration_button.png"></a>
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  </td>
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  <td><em><a href="https://2011.igem.org/Team:Paris_Bettencourt/GFPLac_diffusion">YFP-TetR/TetO array experiment</a></em> This experiment is an improvement of the GFP diffusion experiment of the original paper. To observe significant fluorescence in the neighboring cells, lots of molecules have to pass through the tubes. Using the affinity of the TetR for the TetO array, we want to concentrate in one spot the YFP molecules to better monitor this diffusion.
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<td style="text-align:center;">Concentrator</td>
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<td style="text-align:center;">Yes</td>
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  </td>
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</tr>
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<tr>
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  <td style="width:200px; text-align:center"><a href="https://2011.igem.org/Team:Paris_Bettencourt/ComS_diffusion"><img style="width:150px; margin-top:20px;" src="https://static.igem.org/mediawiki/2011/2/21/ComS-button.png"></a>
 +
  </td>
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  <td><em><a href="https://2011.igem.org/Team:Paris_Bettencourt/ComS_diffusion">ComS diffusion</a></em> ComS is an inhibitor of the MecA protease in <i>B.subtilis</i>. It plays a key role in the triggering of the competence and sporulation mechanisms. The idea is to trigger the switch of the MeKS system of the receptor cell by diffusing ComS through the nanotubes.
 +
  </td>
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<td style="text-align:center;">Switch</td>
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<td style="text-align:center;">Mono-<br/>directional</td>
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</tr>
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</table>
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<h2>Feedback from the models</h2>
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 +
<p>Our <a href="https://2011.igem.org/Team:Paris_Bettencourt/Modeling">modeling</a> showed us which designs would theoritically be the easiest to monitor and which one might only lead to inconclusive results.<p>
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 +
<p>The feedback from our models showed us that:
<ul>
<ul>
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<li>[[Team:Paris_Bettencourt/T7_diffusion|The T7 diffusion:]] The principle of this experiment is to pass T7 polymerase through the nanotubes, this T7 activating the T7 amplifier system in the receptor cell.</li>
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<li><em>Passive diffusion</em> alone can explain the results observed in the Dubey/Ben-Yehuda article, although other processes might contribute significantly</li>
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<li>[[Team:Paris_Bettencourt/Xis_diffusion|The Xis protein diffusion:]] Xis is a small partner of an exisase. The latter will exise a stop codon on the DNA strand that is preventing the expression of the GFP.</li>
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        <li><em>Diffusion times</em> are too short compared to genetic response for us to monitor properly</li>
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<li>[[Team:Paris_Bettencourt/ComS_diffusion|The ComS diffusion:]] The idea is to trigger the switch of the MeKS system by diffusing ComS through the nanotubes. </li>
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<li>All our systems will respond in <em>approximately one hour</em> which fits nicely with the timescale of the GFP diffusion observed in the Dubey/Ben-Yehuda article</li>
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<li>[[Team:Paris_Bettencourt/tRNA_diffusion|The amber suppressor tRNA diffusion:]] The principle of this design is to produce in one cell a amber supressor tRNA that will diffuse through the nanotubes. The receptor cell holds the gene for a T7 with amber stop codons that cannot be translated into a functional protein as long as the tRNA amber suppressor is not present in the cell. Once expressed, the T7amber will trigger the T7 amplification system.</li>
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<li>We know roughly <em>how many molecules will be required to activate</em> each of our system (500 for ComS diffusion, 10 for T7 RNA polymerase, etc.)</li>
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<li>[[Team:Paris_Bettencourt/Xylose_diffusion|The xylose diffusion:]] Xylose accumulated in one of the cell diffuse through the tubes and trigger the amplifier in the receiver cell.</li>
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<li>Since we change experimental conditions for the Coms diffusion system and the Sin Operon system, we know those <em>two systems might not work as expected</em></li>
</ul>
</ul>
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<br>
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</p>
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<p>Using all these modeling results, we were able to <em>design properly our experiments</em> and have some expectations as to the results.</p>
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<h2>Designs for B. Subtilis-E. Coli communication</h2>
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<h2>Others designs not built</h2>
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<p>We want to demonstrate that communication through nanotubes can be established between Gram+ and Gram- bacteria. This would be the first demonstration of protein exchange between species using this newly discovered means of communication. E. Coli will be used as a model for Gram- bacteria. However, we don't know if this communication exists, if it will occur with the periplasm or directly with the cytoplasm. We have two type of designs.</p>
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<p>We had not the manpower to try all the ideas we had. We designed some systems in order to investigate the <i>E. coli / B. subtilis</i> connection nature but had no time to investigate this question further. Nonetheless, we present some of our design ideas for solving this problem in this section.</p>
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<h3>If communication happens with the cytoplasm</h3>
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<p><i>B.subtilis</i> is Gram- so the nanotubes seem to create a link from the cytoplasm of the first cell to the cytoplasm of the second cell. In the case of the Gram negative bacteria, like<i> E.coli</i>, we wonder how the nanotubes connect the cells. Do they create a link with the periplasm or with the cytoplasm? To test the two hypotheses, we tried to create a design for each of the two possibilities. If one work and the other does not, the answer to this question will be known.</p>
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<p>If the communication happens directly with the cytoplasm, we can basically use the same systems used for the Subtilis-Subtilis communication.</p>
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<h3>Design for <i>B.subtilis / E.coli</i> if the connection happens with the cytoplasm</h3>
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<a href="https://2011.igem.org/File:cytoplasm_connection_with_coli.jpg"><img src="../wiki/images/3/3a/Cytoplasm_connection_with_coli.jpg" style="width: 600px; margin-left: 185px;"></a></br>
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[[File:cytoplasm_connection_with_coli.jpg|thumb|center|upright=2.0|Schematics of the communication happening directly to the cytoplasm]]
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<p>If the connection between <i>E.coli</i> and <i>B.subtilis</i> is happening through the cytoplasm, the type of connection could be summed up by the following schematic.</p>
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 +
<img src="../wiki/images/3/3a/Cytoplasm_connection_with_coli.jpg" style="width: 500px; margin-left: 185px;">
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<p><center><b>Schematics of the communication happening directly to the cytoplasm</b></center></p>
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<p>In this case, be can re-use most of the designs of the first section (see the compatibility column). We also have proposed this additional design:</p>
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<p>The compatibility of the different systems will have to verified.</p>
 
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<p>On the other hand, we can re-use their Push-on push-off system of Pekin iGEM team of 2010, adapt it and use it in the receptor cell. The designs are the following:</p>
 
<ul>
<ul>
-
<li>[[Team:Paris_Bettencourt/C1_diffusion|The C1(ind) diffusion:]] The indestructible-C1  diffusion will change the toggle switch state</li>
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<li><a href="https://2011.igem.org/Team:Paris_Bettencourt/Xis_diffusion">Xis protein diffusion</a> Xis is a small partner of an excisase. The latter will excise a stop codon on the DNA strand that is preventing the expression of the GFP. We had no time to build this design, but the idea is summed up <a href="https://2011.igem.org/Team:Paris_Bettencourt/Xis_diffusion">here</a>.</li>
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<li>[[Team:Paris_Bettencourt/RecA_diffusion|The RecA* diffusion:]] The RecA diffusion will help change the toggle switch state</li>
+
</ul>
</ul>
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<h3>If communication happens with the periplasm</h3>
 
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<p>In case the communication happens with the periplasm, we have to think about molecules that can be transported into the cytoplasm.</p>
 
-
<a href="https://2011.igem.org/File:Periplasm_conection.jpg"><img src="../wiki/images/wiki/images/4/40/Periplasm_conection.jpg" style="width: 600px; margin-left: 185px;"></a></br>
 
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[[File:Periplasm_conection.jpg|thumb|center|upright=2.0|Schematic of the communication happening through the periplasm. A special transporter have to be included in the signalling process]]
 
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<p>We have to think about more sophisticated approaches. The designs are as following:</p>
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<h3>Design for <i>B.subtilis / E.coli</i> if the connection happens with the periplasm</h3>
 +
 
 +
<p>In case the communication happens with the periplasm, we had to think about molecules that can be then transported into the cytoplasm.</p>
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 +
<a href="https://2011.igem.org/File:Periplasm_conection.jpg"><img src="https://static.igem.org/mediawiki/2011/4/40/Periplasm_conection.jpg" style="width: 500px; margin-left: 185px;"></a></br>
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<p><center><b>Schematic of the communication happening through the periplasm.</b></center></p>
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 +
<p>We had to design more sophisticated approaches. The ideas are the following:</p>
<ul>
<ul>
-
<li>[[Team:Paris_Bettencourt/MPB_diffusion|The MBP diffusion:]] We need a CRP+, MBP- E.Coli mutant. We produce the MBP protein in Bacillus subtilis and make it diffuse through the nanotubes. As long as the MBP has not reached the E. Coli periplasm, the cell cannot digest the maltose in the medium. The indirect induction of MalR by MBP triggers the expression of the GFP reporter.</li>
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<li><a href="https://2011.igem.org/Team:Paris_Bettencourt/MPB_diffusion">MBP diffusion:</a> we need a CRP+, MBP- <i>E.coli</i> mutant. We produce the MBP protein in <i>B.subtilis</i> and make it diffuse through the nanotubes. As long as the MBP has not reached the periplasm of <i>E.coli</i>, the cell cannot digest the maltose in the medium. The indirect induction of MalR by MBP triggers the expression of the GFP reporter.</li>
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<li>[[Team:Paris_Bettencourt/OmpR diffusion|The OmpR diffusion:]] We need a OmpR- Receptor* E. Coli mutant. We produce the OmpR protein in Bacillus Subtilis. As long as the OmpR has not diffused from B. Subtilis, the signaling cascade cannot be activated. With the rescue by Bacillus Subtilis of the OmpR protein, the expression of the reporter gene is activated.</li>
+
</ul>
</ul>
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<h3>What can we conclude?</h3>
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<br>
 +
 
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<p>If one of these designs works, we would prove that the interspecial nanotube communication exists and would find the eventual location of the connection with the membrane.</p>
 
<br>
<br>
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<h1>Designs for a Master-Slave system</h1>
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<!--<h1>Designs for a Master-Slave system</h1>
-
<p>The principle of Step 2 is to build a Master-Slave system, where the Master controls the state of the Slave cell in a monodirectional exchange.</p>
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<p>The principle of Step 2 is to build a Master-Slave system, where the Master controls the state of the Slave cell in a mono-directional exchange.</p>
<a href="https://2011.igem.org/File:Master_Slave_system_principle.jpeg"><img src="../wiki/images/5/5a/Master_Slave_system_principle.jpeg" style="width: 600px; margin-left: 185px;"></a></br>
<a href="https://2011.igem.org/File:Master_Slave_system_principle.jpeg"><img src="../wiki/images/5/5a/Master_Slave_system_principle.jpeg" style="width: 600px; margin-left: 185px;"></a></br>
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[[File:Master_Slave_system_principle.jpeg|thumb|center|upright=2.0|Summary of the principle of the Master-Slave experiments]]
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<p><center><b>Specification of the master slave design</b></center></p>
<p>Such a design implies the reversibility of all the sub-systems, the activators and the amplifiers in particular.</p>
<p>Such a design implies the reversibility of all the sub-systems, the activators and the amplifiers in particular.</p>
Line 93: Line 201:
<p>Several potential designs are summed-up below:</p>
<p>Several potential designs are summed-up below:</p>
<ul>
<ul>
-
<li>[[Team:Paris_Bettencourt/PonPoff_system|The diffusive RecA push-on push-off system:]] In this design we re-use the 2010 Pekin iGEM team's push-on push-off system, but instead of triggering the change by UV, we trigger it with a constitutively active RecA mutant that we diffuse through the nanotubes. We want the emitter cell (the master cell) to control the toggle switch state in the slave cell.</li>
+
<li><a href="https://2011.igem.org/Team:Paris_Bettencourt/ComS_diffusion">The ComS system:</a> The characterization step of the MeKS system turn to be reversible. It can be considered as well as a Master/Slave system.</li>
-
<li>[[Team:Paris_Bettencourt/ComS_diffusion|The ComS system:]] The caracterisation step of the MeKS system turn to be reversible. It can be considered as well as a Master/Slave system.</li>
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</ul>-->
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</ul>
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<br>
<br>
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<h1>Designs for a bidirectional communication</h1>
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<div id="citation_box">
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<p id="references">References</p>
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<p>If we succeed in establishing a monodirectional communication, we may go on and try to build a bidirectional communication system. Here is the general draft:</p>
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<ol>
-
<a href="https://2011.igem.org/File:Principle_of_bidirectional_communication.jpg"><img src="../wiki/images/0/00/Principle_of_bidirectional_communication.jpg" style="width: 600px; margin-left: 185px;"></a></br>
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<li><i>Intercellular Nanotubes Mediate Bacterial Communication</i>, Dubey and Ben-Yehuda, Cell, available <a href="http://bms.ucsf.edu/sites/ucsf-bms.ixm.ca/files/marjordan_06022011.pdf">here</a></li>
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[[File:Principle_of_bidirectional_communication.jpg|thumb|center|upright=2.0|Summary of the principle of the bidirectional cummunication]]
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<p>The genetic design combines the two previously described amplification systems. The complete sum-up is the following:</p>
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[[File:General_Scheme_for_bidirectional_communication.jpg|thumb|center|upright=3.0|Complete design]]
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Latest revision as of 01:51, 29 October 2011

Team IGEM Paris 2011

Designs overview

Introduction

Our goal for this summer is to characterize the travel of various molecules through nanotubes. In order to do so, we developed several designs. In this page, we explain the principle of the different designs we have built.

In the original paper [1], the authors directly observed the passage of molecules through the nanotubes. The idea behind our approach is to use synthetic biology methods to improve the sensitivity and the resolution of these experiments. Relying on signal amplification and natural, as well as artificial, bistable switches, we want to detect, with a resolution at the molecular level, the passage from one emitter cell to the receiver.

our logo

Main questions

The nanotube discovery is very recent. Lots of questions remain unsolved concerning the mechanism behind the formation of the tubes, the efficiency of the transfer, the extent of the process. The existence of the tubes themselves remains a subject of controversy within the scientific community.

We aim to bring additional proof of their existence and better characterize the extent of this phenomenon. Here is the list of the questions we have been asking ourselves, and that our designs aim to answer:

  • What is the nature of the process? (passive or active transport)
  • What kinds of molecules can pass through the nanotubes?
  • Does the communication happen only between B. subtilis? Or can it also exist between different species?
  • If a communication can be established with Gram negative bacteria, is the communication happening through the periplasm or the cytoplasm?

There is also the question of the formation of the tubes. However, this specific problem would require a lot more than a summer and a team of undergraduates to be fully investigated. We therefore choose not to focus too much on it, even though we have some ideas.

Specification of our designs

We started designing the project with these previously stated questions in mind. Using several molecules of different sizes and nature, from a T7 RNA polymerase to ComS, we aim to test the nature, size and number of the molecules we want to diffuse through the nanotubes.

We decided that our constructs should follow the global idea summed up in the following scheme:


General plan for the experiment design to characterize the nanotubes

As explained in our modeling section, diffusion seems to be too quick comparatively to genetic networks response time to be accurately measured. We therefore focused on testing the nature, the number and the size of the molecules we try to pass trough the nanotubes.

In B.subtilis, nanotubes are reported allow transfer to the cytoplasm. We had to find molecules that can be expressed in the first cell and then trigger a genetic circuit in the second cell. The image below illustrates the idea:


Schematic of the supposed connection between the cells via the nanotubes

Genetic devices we tested

Using these specifications, we designed several sensitive genetic circuits that can be trigerred by molecules of various sizes. The molecules chosen cover 1 orders of magnitude of radius.





Description Type of switch B. subilis/
E. coli
compatibility
T7 polymerase diffusion The T7 RNA polymerase is the RNA polymerase of the T7 phage. This is a big molecule, that recognizes a very specific promoter orthogonal to B.subtilis. Irreversible
amplification
Yes
Amber suppressor tRNA diffusion The principle of this design is to produce in one cell an amber supressor tRNA that will diffuse through the nanotubes. The receptor cell holds the gene for T7 with amber stop codons that cannot be translated into a functional protein as long as the tRNA amber suppressor is not present in the cell. Once expressed, the functional amber T7 RNA polymerase will trigger the T7 amplification system. Irreversible
amplification
Yes
KinA diffusion The Sin operon system controls the sporulation switch of B. subtilis. Making it diffuse from a non sporulating strain to a receiver cell makes the receiver cell to sporulate. We also use fluorescent sporulation reporters to monitor the experiment properly. Switch Mono-
directional
Lambda switch design The CI is a regulation protein from the lambda phage switch. Make it diffuse would change the state of the toogle switch from the push-on push-off system, from the PKU 2007. Switch Mono-
directional
YFP-TetR/TetO array experiment This experiment is an improvement of the GFP diffusion experiment of the original paper. To observe significant fluorescence in the neighboring cells, lots of molecules have to pass through the tubes. Using the affinity of the TetR for the TetO array, we want to concentrate in one spot the YFP molecules to better monitor this diffusion. Concentrator Yes
ComS diffusion ComS is an inhibitor of the MecA protease in B.subtilis. It plays a key role in the triggering of the competence and sporulation mechanisms. The idea is to trigger the switch of the MeKS system of the receptor cell by diffusing ComS through the nanotubes. Switch Mono-
directional

Feedback from the models

Our modeling showed us which designs would theoritically be the easiest to monitor and which one might only lead to inconclusive results.

The feedback from our models showed us that:

  • Passive diffusion alone can explain the results observed in the Dubey/Ben-Yehuda article, although other processes might contribute significantly
  • Diffusion times are too short compared to genetic response for us to monitor properly
  • All our systems will respond in approximately one hour which fits nicely with the timescale of the GFP diffusion observed in the Dubey/Ben-Yehuda article
  • We know roughly how many molecules will be required to activate each of our system (500 for ComS diffusion, 10 for T7 RNA polymerase, etc.)
  • Since we change experimental conditions for the Coms diffusion system and the Sin Operon system, we know those two systems might not work as expected

Using all these modeling results, we were able to design properly our experiments and have some expectations as to the results.

Others designs not built

We had not the manpower to try all the ideas we had. We designed some systems in order to investigate the E. coli / B. subtilis connection nature but had no time to investigate this question further. Nonetheless, we present some of our design ideas for solving this problem in this section.

B.subtilis is Gram- so the nanotubes seem to create a link from the cytoplasm of the first cell to the cytoplasm of the second cell. In the case of the Gram negative bacteria, like E.coli, we wonder how the nanotubes connect the cells. Do they create a link with the periplasm or with the cytoplasm? To test the two hypotheses, we tried to create a design for each of the two possibilities. If one work and the other does not, the answer to this question will be known.

Design for B.subtilis / E.coli if the connection happens with the cytoplasm

If the connection between E.coli and B.subtilis is happening through the cytoplasm, the type of connection could be summed up by the following schematic.

Schematics of the communication happening directly to the cytoplasm

In this case, be can re-use most of the designs of the first section (see the compatibility column). We also have proposed this additional design:

  • Xis protein diffusion Xis is a small partner of an excisase. The latter will excise a stop codon on the DNA strand that is preventing the expression of the GFP. We had no time to build this design, but the idea is summed up here.

Design for B.subtilis / E.coli if the connection happens with the periplasm

In case the communication happens with the periplasm, we had to think about molecules that can be then transported into the cytoplasm.


Schematic of the communication happening through the periplasm.

We had to design more sophisticated approaches. The ideas are the following:

  • MBP diffusion: we need a CRP+, MBP- E.coli mutant. We produce the MBP protein in B.subtilis and make it diffuse through the nanotubes. As long as the MBP has not reached the periplasm of E.coli, the cell cannot digest the maltose in the medium. The indirect induction of MalR by MBP triggers the expression of the GFP reporter.



References

  1. Intercellular Nanotubes Mediate Bacterial Communication, Dubey and Ben-Yehuda, Cell, available here