Latest revision as of 02:28, 29 October 2011

Team IGEM Paris 2011

Achievements

Preliminary experiments

In the Dubey and Ben-Yehuda paper [1] a set of simple experiments are presented in support of the existence of the nanotubes. We reproduced some of them in order to demonstrate the existence of these entities as a medium of communication between bacteria, and to be sure we are in the good experimental conditions to produce them when working with our detectors (see below).

We reproduced the two keystone experiments of the paper: the GFP diffusion, and the antibiotic resistance exchange, where our results point to an alternative explanation.

The GFP diffusion experiment is a simple experiment. One Bacillus subtlis strain producing GFP is mixed with a wild type strain. If nanotubes are formed, the GFP would diffuse through the tubes and color the non fluorescent strain. We succeded in reproducing the results of the paper, showing GFP diffusion when gfp+ cells are close to gfp- cells. We invite you to visit corresponding the page to learn more about what we did and the results we had.

The antibiotic resistance exchange is a more tricky experiment in which bacteria are shown to exchange resistance enzymes through nanotube and allow the population to survive even though all the cells does not carry the resistance gene. We invite you to visit corresponding the page to learn more about what we did and the results we obtained that offer an alternative simple explanation that does not evoke use of nanotubes.

Testing for the presence of nanotubes

The goal of all our designs was to test for the presence of and characterize nanotubes. You will find here the experiments we conducted to this end, carefully designed according to the results of our computational modeling.

The YFP concentrator This design relies on a TetO-array which allow us to concentrate YFP-TetR fusion proteins. We were able to to a E.coli to B.subtilis diffusion through nanotubes experiment with this design. Preliminary results with coli-subtilis did not show any transfer in microscopy experiments.

T7 RNA polymerase diffusion In this design, we introduce the use of the T7 polymerase both as the transfer molecule and as the auto-amplification system. We were able to do E.coli to B.subtilis as well as B.subtilis-B.subtilis diffusion through nanotubes experiment with this design. We also tried to experiment with our microfluidic chip with this design. Preliminary results with coli-subtilis or subtilis-subtilis did not show any transfer in microscopy experiments.

Building and characterizing new devices

Before testing the nanotubes, we had to design entirely these new devices. They are composed of an emitter, a receptor and an amplifier subunit.

	The YFP concentrator This design relies on a TetO-array which allow us to concentrate YFP-TetR fusion proteins. We were able to properly characterize it through microscopy images.
	T7 RNA polymerase diffusion In this design, we introduce the use of the T7 polymerase both as the transfer molecule and as the auto-amplification system. We were able to properly characterize it through microscopy images and 96-well kinetics experiments.
	tRNA amber diffusion The tRNA amber is the smallest molecule we are trying to get pass the nanotubes . At the receiving end: T7 polymerase with two modified amber codons coupled with the T7 auto-amplifier and T7-driven GFP expression. We have also characterized the pHyperSpank promoter. We were able to properly characterize it through microscopy images and 96-well kinetics experiments.

Using bistable switches

During our brainstormings, we noticed several natural or artificial bistable switches that could serve both as a receptor and an auto-amplifier. One molecule carefully chosen could toggle the switch in another position. All we have to do is see if it diffuses through the nanotubes.

	ComS diffusion We took advantage of a switch already existing in B .subtilis (the ComK/ComS switch) and tried to see if we could toggle it from one state to the other using molecules diffusing through the nanotubes.
	Sin Operon We took advantage of a switch already existing in B.Subtilis (the Sin operon switch regulating sporulation and biofilm formation) and tried to see if we could toggle it from one state to the other using molecules diffusing through the nanotubes.
	Lambda switch We took advantage of an artificial switch in E.coli created by the PKU team of 2007. We tried to see if we could toggle it from one state to the other using molecules diffusing through the nanotubes.

References

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

@@ Line 1: / Line 1: @@
 {{:Team:Paris_Bettencourt/tpl_test}}
 <html>
-<h1>Designs overview</h1>
-<h2>Introduction</h2>
+<h1>Achievements</h1>
+<h2>Preliminary experiments</h2>
+<p>In the Dubey and Ben-Yehuda paper <a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/List#references">[1]</a> a set of simple experiments are presented in support of the existence of the nanotubes. We reproduced some of them in order to demonstrate the existence of these entities as a medium of communication between bacteria, and to be sure we are in the good experimental conditions to produce them when working with our detectors (see below).</p>
+<p>We reproduced the two keystone experiments of the paper: the GFP diffusion, and the antibiotic resistance exchange, where our results point to an alternative explanation.</p>
 <table>
 <tr>
-    <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>
+  <td style="width:200px; text-align:center"><a href="https://2011.igem.org/Team:Paris_Bettencourt/GFP_diff"><img style="width:150px" src="https://static.igem.org/mediawiki/2011/8/80/GFP-diff-button.png"></a>
-<td>
+  </td>
-<p><b>Our goal for this summer is characterizing the exchange of proteins through nanotubes as best as we can. In order to do that, we want to construct several design. In this page, we are going to explain you the principle of the different designs we have builded.</b></p>
+  <td><b>The <a href="https://2011.igem.org/Team:Paris_Bettencourt/GFP_diff">GFP diffusion experiment</a></b> is a simple experiment. One Bacillus subtlis strain producing GFP is mixed with a wild type strain. If nanotubes are formed, the GFP would diffuse through the tubes and color the non fluorescent strain. We succeded in reproducing the results of the paper, showing GFP diffusion when <i>gfp+</i> cells are close to <i>gfp-</i> cells. We invite you to <a href="https://2011.igem.org/Team:Paris_Bettencourt/GFP_diff">visit corresponding the page</a> to learn more about what we did and the results we had.
+  </td>
-<p>In the original paper, the author observe directly the molecules that passes through the nanotubes. The idea behind our approach is to use <em>synthetic biology methods</em> to go farther on the sensitivity and the resolution of these experiments. Relying on <em>signal amplification</em> methods and natural and artificial <em>bistable switches</em> to detect at the molecule resolution when a few molecules have diffused from <b>one emittor cell</b> to <b>the receiver</b>.</p>
+</tr>
-</td>
+<tr>
+  <td style="width:200px; text-align:center"><a href="https://2011.igem.org/Team:Paris_Bettencourt/Atb_exp"><img style="width:150px" src="https://static.igem.org/mediawiki/2011/2/25/Atb_button.png"></a>
+  </td>
+  <td><p><b>The <a href="https://2011.igem.org/Team:Paris_Bettencourt/Atb_exp">antibiotic resistance exchange</a></b> is a more tricky experiment in which bacteria are shown to exchange resistance enzymes through nanotube and allow the population to survive even though all the cells does not carry the resistance gene. We invite you to <a href="https://2011.igem.org/Team:Paris_Bettencourt/Atb_exp">visit corresponding the page</a> to learn more about what we did and the results we obtained that offer an alternative simple explanation that does not evoke use of nanotubes.</p>
+  </td>
 </tr>
 </table>
+<h2>Testing for the presence of nanotubes</h2>
-<h2>Main questions behinds</h2>
+<p>The goal of all our designs was to <em>test for the presence of and characterize nanotubes</em>. You will find here the experiments we conducted to this end, carefully designed according to the results of our <a href="https://2011.igem.org/Team:Paris_Bettencourt/Modeling">computational modeling</a>.</p>
-<p>The nanotube discorevy is very recent. Though, lots of question remains unsolved, on the processes of <em>the formation of the tubes</em>, the efficiency of the communication, the extent of the process. The existence itself remains contested by some scientists.</p>
-<p>We aim to add additionnal proof of their existence and increase the number of the nanotubes, and characterize better the extent of the phenomenon. Here is the list of the question we are asking ourself, and that our designs aimed to answers:<p>
 <table>
 <tr>
-<td style="width:200px; text-align:center"><img style="width:150px" src="https://static.igem.org/mediawiki/2011/2/2b/Question_mark_button.png">
+  <td style="width:200px; text-align:center"><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/YFP_TetR_diffusion_experiments"><img style="width:150px" src="https://static.igem.org/mediawiki/2011/2/2d/Yfp_diff_button_pb.png"></a>
+  </td>
+  <td><b><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/YFP_TetR_diffusion_experiments">The YFP concentrator</a></b> This design relies on a TetO-array which allow us to concentrate YFP-TetR fusion proteins. We were able to to a <em><i>E.coli</i> to <i>B.subtilis</i> diffusion through nanotubes experiment</em> with this design. Preliminary results with <i>coli-subtilis</i> did not show any transfer in microscopy experiments.
+  </td>
+</tr>
+<tr>
+  <td style="width:200px; text-align:center;"><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/T7_diffusion_experiments"><img style="width:150px; margin-top:20px;" src="https://static.igem.org/mediawiki/2011/a/a8/T7_diff_button_pb.png"></a>
+  </td>
+  <td><b><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/T7_diffusion_experiments">T7 RNA polymerase diffusion</a></b> In this design, we introduce the use of the T7 polymerase both as the transfer molecule and as the auto-amplification system. We were able to do <em><i>E.coli</i> to <i>B.subtilis</i> as well as <i>B.subtilis-B.subtilis</i> diffusion through nanotubes experiment</em> with this design. We also tried to experiment with our microfluidic chip with this design. Preliminary results with <i>coli-subtilis</i> or <i>subtilis-subtilis</i> did not show any transfer in microscopy experiments.
    </td>
-<td>
-<ul>
-<li><em>How</em> the tubes are forming?</li>
-<li>What is the <em>speed</em> of the process? (passive or active transport)</li>
-<li>Can we pass all kind of molecules?</li>
-<li>Is the communication happens only in between B. subtilis, or can it happens also interspecies or even between E. coli strains?</li>
-<li>If a communication is established with a Gram positive bacteria, is the communication happening through the periplasm of the cytoplasm?</li>
-</ul>
-</td>
 </tr>
 </table>
-<h2>Specification of our designs</h2>
-<p>We started designing the project with these the previously stated questions in questions in mind. Using several molecules of <em>different sizes</em>, from a T7 polymerase to a tRNA amber supressor, we aim to test the capability and size limit for the molecules that can pass through the tubes. But, if the diffusion is passive, the speed of diffusion should be proportional to the diffusion coefficient D, that is to say inversely proportional to the radius of the object. Can we design a system fast-responding enough that would allow us to measure this speed of the transfert?</p>
-</p>We decided that our constructs should follow the global idea summed up in the following picture:</p>
-<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>
-<p><center><b><u>Fig1:</u> General plan for the experiment design to characterize the nanotubes</center></b></p>
-<p>The problem is that each design needs a different couple of signal emitter/receptor. However, even with good models, the response time cannot be predicted easily because little is known about the transfer through the nanotubes. We tried to limit the impact of this issue by the following measures:</p>
-<p>
-<ul>
-<li>Use each time the same actuator and the same monitor to have comparable response times in the different systems.</li>
-<li>The relevant measure is not the response time itself, but the increase of response time when the two constructs are in the same cell or in different cells linked by a nanotube</li>
-</ul>
-</p>
-<p>The idea is if we can measure this increase of time for different sized, that is to say of different D coefficien, of the molecule we should get a relation in lambda/D, the diffusion is an active process. Else, the process is active. See the <a href="https://2011.igem.org/Team:Paris_Bettencourt/Hypothesis">modeling pages</a> for more details about this assuption.</p>
-<br>
-<p>In <i>B.subtilis</i>, nanotubes are reported to be formed by the cytoplasm. We had to find a molecule that can be expressed in the first cell that can trigger a genetic circuit in the second cell. The image below illustrates the idea:</p>
-<a href="https://2011.igem.org/File:cytoplasm_cytoplasm_communication.jpg"><img src="../wiki/images/4/49/Cytoplasm_cytoplasm_communication.jpg" style="width: 500px; margin-left: 185px;"></a></br>
-<p><center><b><u>Fig2:</u> Schematic of the supposed connection between the cells via the nanotubes</b></center></p>
-<h2>Building fast new genetic devices</h2>
-<p>Using these specification, we designed several sensitive genetic circuit that can be trigerred by molecules of various size. The molecules chosen cover 2 orders of magnitude of Radius. Here, they are classified from the biggest to the smallest.</p>
+<h2>Building and characterizing new devices</h2>
+<p>Before testing the nanotubes, we had to design entirely these new devices. They are composed of an emitter, a receptor and an amplifier subunit.</p>
 <table>
 <tr>
-<td></td>
+   <td style="width:200px; text-align:center"><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/YFP_TetR_diffusion"><img style="width:150px" src="https://static.igem.org/mediawiki/2011/d/d0/YFP_concentration_button.png"></a>
-<td></td>
-<td><b><u>Type of switch</u></b></td>
-<td><b><u>B. subilis<br/>E. coli<br/>compatibility</u></b></td>
-</tr>
-<tr>
-   <td style="width:200px; 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>
    </td>
-   <td><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>.
+   <td><b><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/YFP_TetR_diffusion">The YFP concentrator</a></b> This design relies on a TetO-array which allow us to concentrate YFP-TetR fusion proteins. We were able to properly characterize it through microscopy images.
    </td>
-<td>Irreversible<br/>amplification</td>
-<td>Yes</td>
 </tr>
 <tr>
-   <td style="width:200px; 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>
+   <td style="width:200px; text-align:center;"><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/T7_diffusion"><img style="width:150px; margin-top:20px;" src="https://static.igem.org/mediawiki/2011/e/e4/T7_button.png"></a>
    </td>
-   <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><b><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/T7_diffusion">T7 RNA polymerase diffusion</a></b> In this design, we introduce the use of the T7 polymerase both as the transfer molecule and as the auto-amplification system.  We were able to properly characterize it through microscopy images and 96-well kinetics experiments.
    </td>
-<td>Irreversible<br/>amplification</td>
-<td>Yes</td>
 </tr>
 <tr>
-   <td style="width:200px; 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/2011/2/21/ComS-button.png"></a>
+   <td style="width:200px; text-align:center"><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/tRNA_diffusion"><img style="width:150px; margin-top:20px;" src="https://static.igem.org/mediawiki/2011/5/53/TRNAamber-button.png"></a>
    </td>
-   <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 B. subtilis. Making it diffuse from a non sporulating strain to a receiver cell makes the receiver cell to sporulate.
+   <td><b><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/tRNA_diffusion">tRNA amber diffusion</a></b> The tRNA amber is the smallest molecule we are trying to get pass the nanotubes . At the receiving end: T7 polymerase with two modified amber codons coupled with the T7 auto-amplifier and T7-driven GFP expression. We have also <a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/pHyperSpank">characterized the pHyperSpank promoter</a>. We were able to properly characterize it through microscopy images and 96-well kinetics experiments.
    </td>
-<td>Switch</td>
-<td>Mono-<br/>directional</td>
 </tr>
+</table>
+<h2>Using bistable switches</h2>
+<p>During our brainstormings, we noticed several natural or artificial bistable switches that could serve both as a receptor and an auto-amplifier. One molecule carefully chosen could toggle the switch in another position. All we have to do is see if it diffuses through the nanotubes.
+<table>
 <tr>
-   <td style="width:200px; text-align:center">Lambda switch<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/2011/2/21/ComS-button.png"></a>
+   <td style="width:200px; text-align:center"><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/ComS_diffusion"><img style="width:150px; margin-top:20px;" src="https://static.igem.org/mediawiki/2011/2/21/ComS-button.png"></a>
    </td>
-   <td><em><a href="https://2011.igem.org/Team:Paris_Bettencourt/SinOp">CI diffusion:</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 PKU 2007.
+   <td><b><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/ComS_diffusion">ComS diffusion</a></b> We took advantage of a switch already existing in <i>B .subtilis</i> (the ComK/ComS switch) and tried to see if we could toggle it from one state to the other using molecules diffusing through the nanotubes.
    </td>
-<td>Switch</td>
-<td>Mono-<br/>directional</td>
 </tr>
 <tr>
-   <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>
+   <td style="width:200px; text-align:center"><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/SinOp"><img style="width:150px; margin-top:20px;" src="https://static.igem.org/mediawiki/2011/a/aa/SinOp-button.png"></a>
    </td>
-   <td><em><a href="https://2011.igem.org/Team:Paris_Bettencourt/GFPLac_diffusion">The YFP-TetR/TetO array experiment:</a></em> This experiment is an improvement of the previous one. To observe significant fluorescence in the neighboring cell, lots of molecules have to pass through the tubes. Using the affinity of the TetR for the TetO array, we want to concentrate at one point the YFP molecules to better observe this diffusion.
+   <td><b><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/SinOp">Sin Operon</a></b> We took advantage of a switch already existing in <i>B.Subtilis</i> (the Sin operon switch regulating sporulation and  biofilm formation) and tried to see if we could toggle it from one state to the other using molecules diffusing through the nanotubes.
-<td>-</td>
-<td>Yes</td>
    </td>
 </tr>
 <tr>
-   <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 style="width:200px; 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>
    </td>
-   <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. A major precaution is to prevent the first cell from sporulating.
+   <td><b><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/Lambda_switch">Lambda switch</a></b>  We took advantage of an artificial switch in <i>E.coli</i> created by the <a href="https://2007.igem.org/Peking">PKU team of 2007</a>. We tried to see if we could toggle it from one state to the other using molecules diffusing through the nanotubes.
    </td>
-<td>Switch</td>
-<td>Mono-<br/>directional</td>
 </tr>
 </table>
+<div id="citation_box">
+<p id="references">References</p>
+<ol>
-<h2>Others designs we had no time to build</h2>
+<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>
+</ol>
-<p>We neither had time nor the manpower to try all the ideas we had. Some designs too complicated to be feasible. We also wanted to investigate at length the <i>Subtilis/Coli</i> connexions but had no time to do it properly. We nonetheless present all our ideas in this section.</p>
+</div>
-<h3>Abandonned design for <i>B.subtilis</i></h3>
-<ul>
-<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 on the linked page.</li>
-</ul>
-<h3>Designs for <i>B.subtilis-E.coli</i> communication</h3>
-<p>The article demonstrates that nanotubes established inside the <i>B.subtilis</i> family  can also be formed between <i>B.subtilis</i> and <i>E.coli</i>. These two types of bacteria are really different since one is Gram+ and the other is Gram-. Their membrane is really different since there is no periplasm in <i>B.subtilis</i>. The existence of <em>inter-species nanotubes</em> did really spike our interest and we also created designs to test this aspect of the nanotube communication. We had few time to test these ideas but we present here the designs we thought of. For all the designs which are simply the ones for <i>B.subtilis</i> applied to a <i>Subtilis/Coli</i> connexion, we plan to experiment them in the future since they require very little additional work.</p>
-<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+ bacteria, like<i> E.coli</i>, we wonder which membrane forms the nanotubes . 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>
-<p>If nanotube communication can be established between <i>E.coli</i> and <i>B.subtilis</i>, it is not known whether the  communication happens through the cytoplasm or the periplasm. To test these two hypotheses, two designs were made:</p>
-<h4>If communication happens with the cytoplasm</h4>
-<p>The image below sums up the kind of connection that can be established if nanotubes connect the two cytoplasms. Hence, some of the designs we have shown above are still available for <i>B.subtilis</i> to <i>E.coli</i>.</p>
-<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: 500px; margin-left: 185px;"></a></br>
-<p><center><b><u>Fig3:</u> Schematics of the communication happening directly to the cytoplasm</b></center></p>
-<p>Here are the designs created in this case.</p>
-<ul>
-<li><a href="https://2011.igem.org/Team:Paris_Bettencourt/T7_diffusion">T7 polymerase diffusion:</a> The T7 RNA polymerase is the RNA polymerase of the T7 phage. This is a pretty big molecule, that recognizes a very specific promoter orthogonal to <i>B.subtilis</i> and <i>E.coli</i>.</li>
-<li><a href="https://2011.igem.org/Team:Paris_Bettencourt/SinOp">KinA diffusion:</a> The idea is to make a complex partner to diffuse from one cell to the other in order to trigger the sporulation of the second cell, even in exponential phase.</li>
-<li><a href="https://2011.igem.org/Team:Paris_Bettencourt/ComS_diffusion">ComS diffusion:</a> 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. A major precaution is to prevent the first cell from sporulating.</li>
-<li><a href="https://2011.igem.org/Team:Paris_Bettencourt/tRNA_diffusion">Amber suppressor tRNA diffusion:</a> The principle of this design is to produce in one cell an amber suppressor 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 functional amber T7 RNA polymerase will trigger the T7 amplification system.</li>
-</ul>
 <br>
-<h4>If communication happens with the periplasm</h4>
-<p>In case the communication happens with the periplasm, we have to think about molecules that can be then transported into the cytoplasm.</p>
-<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>
-<p><center><b><u>Fig4:</u> Schematic of the communication happening through the periplasm.</b></center></p>
- <p>We had to design more sophisticated approaches. The ideas are the following:</p>
-<ul>
-<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>
-<li><a href="https://2011.igem.org/Team:Paris_Bettencourt/OmpR diffusion">OmpR diffusion:</a> we need a OmpR- Receptor* <i>E.coli</i> mutant. We produce the OmpR protein in <i>B.subtilis</i>. As long as the OmpR has not diffused from <i>B.subtilis</i>, the signaling cascade cannot be activated. With the rescue by <i>B.subtilis</i> of the OmpR protein, the expression of the reporter gene is activated.</li>
-</ul>
-<br>
-<p>From electronic microscopy images, the author shows that the communication through the nanotubes can also occurs inter bacteria species. We also designed our experiments so that we can support the existence these interspecies communication with optical microscopy experiments, by placing our emitters and receivers either into B. subtilis and E. coli.</p>
-<h2>Can we conclude on these designs?</h2>
-<p>If one of these designs works, it will be proved that the inter-species nanotube communication exists and this will eventually show location of the connection with the membrane. There was limited time to test these designs but we propose them for future iGEM teams to investigate.</p>
-<br>
-<!--<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 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>
-<p><center><b><u>Fig5:</u> 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>Several potential designs are summed-up below:</p>
-<ul>
-<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>
-</ul>-->
-<br>
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Team:Paris Bettencourt/Experiments/List

From 2011.igem.org