Team:Paris Bettencourt/Project

From 2011.igem.org

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<h1>Designs overview</h1>
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<h2>Introduction</h2>
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<h1>Overview of the project</h1>
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<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>
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<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>
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<h2>Main questions behinds</h2>
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<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>
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<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>
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<p>Mankind is only beginning to grasp the complexity of living organisms. New discoveries often challenge our understanding of life. We believe that synthetic biology can be used as a powerful and reliable tool to help us comprehend and characterize the phenomena we just encountered.</p>
 
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<p>As an iGEM team, we decided to work on one of the most intriguing microbiology discovery of the last decade: the existence of <em>nanotubes communication routes</em> in <i>Bacillus subtilis</i>!</p>
 
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<p>The recent discovery of nanotubes between individual <i>Bacillus subtilis</i> by Dubey and Ben-Yehuda spiked our interest. Through very detailed and advanced microscopy, they showed nanotubes forming between cells and that a wide range of proteins could pass through this communication channel (GFP, calcein, antibiotics, ...). They also showed signs of communication between <i>B.subtilis</i> and <i>E.coli</i>, an entirely different species. This counter-intuitive communication channel could very well have a tremendous impact on evolution, with two different species sharing proteins and even genetic material.</p>
 
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<center><a href="https://2011.igem.org/File:Nanotube_and_GFP.jpeg"><img src="../wiki/images/e/ed/Nanotube_and_GFP.jpeg" style="width:70%; margin: 15px 15px 0px 15px;"></a></center>
 
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<p><b><center><u>Fig1:</u> <i>B.subtilis</i> exchanging molecules through nanotubes network</center></b></p>
 
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<h4>Summary of the article:</h4>
 
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<p>The article published by Dubey and Ben-Yehuda <a href="https://2011.igem.org/Team:Paris_Bettencourt/Project#references">[1]</a> in the Journal <i>Cell</i> is the starting point of our project. In this paper, they show an extraordinary new form of communication between <i>Bacillus subtilis</i> cells and even exchanges with <i>E. coli</i> </p>
 
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<div style="margin-left:50px; margin-right:50px; padding: 5px; border:2px solid black;"><b><p>The article in <em>5 bullet points</em> (all of this happens on solid medium only):
 
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    <li>GFP and calcein, two molecules which can not leave cytoplasm, can be transfered to neighbouring cells in <i>B.subtilis</i>.</li>
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<li><em>How</em> the tubes are forming?</li>
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    <li>A nanotube network can be observed through electronic microscopy between <i>B.subtilis</i> cells.</li>
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<li>What is the <em>speed</em> of the process? (passive or active transport)</li>
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    <li>GFP can be observed passing through these nanotubes.</li>
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<li>Can we pass all kind of molecules?</li>
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    <li>Antibiotic resistance can be transfered between <i>B.subtilis</i> cells or between <i>B.subtilis</i> and <i>E.coli</i>, both in a hereditary and a non-hereditary way.</li>
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<li>Is the communication happens only in between B. subtilis, or can it happens also interspecies or even between E. coli strains?</li>
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    <li>Nanotubes connecting different species (<i>B.subtilis</i>, <i>E.coli</i> and <i>S.aureus</i>) have been oberved with electronic microscopy.</li>
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<li>If a communication is established with a Gram positive bacteria, is the communication happening through the periplasm of the cytoplasm?</li>
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</ul></p></b></div>
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</ul>
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<div style="float:left; width: 250px; margin: 15px 15px 0px 15px;"><a href="https://2011.igem.org/Team:Paris_Bettencourt/GFP_microscopy_Dubey_Ben-Yehuda"><img src="https://static.igem.org/mediawiki/2011/e/e0/BY_fluo_gfp_subt.png" style="width:100%;"></a>
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<h2>Specification of our designs</h2>
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<p><b><center><u>Fig2:</u> Visualizing a Molecular Gradient between Neighboring <i>Bacillus subtilis</i> Cells  (from the Dubey-Ben-Yehuda article) <a href="https://2011.igem.org/Team:Paris_Bettencourt/Project#references">[1]</a></center></b></p></div>
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<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>
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</p>We decided that our constructs should follow the global idea summed up in the following picture:</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><center><b><u>Fig1:</u> General plan for the experiment design to characterize the nanotubes</center></b></p>
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<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>
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The starting point of this paper was the culture of two different strains of <i>B.subtilis</i>. One produces GFP, a fluorescent protein and the other does not. When grown close together on a solid medium, a <em>transfer of fluorescence</em> from the <i>gfp+</i> towards the <i>gfp-</i> neighbouring cells was observed. Interestingly, this transfer was clearly linked to the distance between two different individuals.
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<li>Use each time the same actuator and the same monitor to have comparable response times in the different systems.</li>
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<p>To test if this transfer could be reproduced with smaller molecules, the Dubey/Ben-Yehuda team worked with calcein. Calcein is much smaller than GFP (623 Da to compare to 27kDa). Calcein can be used to label cells as it easily enters the cell but does not leave the cytoplasm afterwards. In addition calcein can be hydrolysed by <i>B.subtilis</i>, resulting in a strong fluorescence. Calcein-free cells growing on a solid medium near calcein-labeled cells exhibited the same behaviour as in the GFP experiment above. Non-fluorescent cells exhibited fluorescence and calcein-labeled cells were less fluorescent as time passed. Controls however showed that <i>calcein+</i> cells kept a steady fluorescence and <i>calcein-</i> cells were not fluorescent.
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<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>
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<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>
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<p> These two experiments suggested that a <em>cell-to-cell close range communication pathway</em> exists in <i>B.subtilis</i>. The Dubey/Ben-Yehuda team investigated this discovery further using electronic microscopy.</p>  
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<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>
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<div style="float:right; width: 300px; margin: 15px 15px 0px 15px;"><a href="https://2011.igem.org/Team:Paris_Bettencourt/Electronic_microscopy_Dubey_Ben-Yehuda"><img src="https://static.igem.org/mediawiki/2011/f/f6/BY_electronic.png" style="width:100%;"></a>
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<p><b><center><u>Fig3:</u> Electronic microscopy from the Dubey-Ben-Yehuda article <a href="https://2011.igem.org/Team:Paris_Bettencourt/Project#references">[1]</a></center></b></p></div>
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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: 500px; margin-left: 185px;"></a></br>
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<p>The pictures support the existence of numerous nanotubes connecting cells. To ensure that these nanotubes could be a significant transfer mechanism, another GFP experiment was tried. Similar to the first one, two antibodies were added. One was an anti-GFP antibody, attaching to the GFP molecules. The other was a secondary antibody attaching to the first one and gold-conjugated. This way, individual GFP molecules could be tagged with the gold-conjugated antibody and followed by electronic microscopy. In this case, they observed <em>GFP molecules moving in the nanotubes</em> from one cell to another.</p>
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<p><center><b><u>Fig2:</u> Schematic of the supposed connection between the cells via the nanotubes</b></center></p>
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<p>The images showed that nanotubes were between <em>30 and 130 nm wide</em> and up to <em>1 &micro;m long</em></p>
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<h2>Building fast new genetic devices</h2>
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<p>The next step was to study <em>antibiotic resistance transfer</em>. Trying to see if non-hereditary (through resistance protein sharing) and hereditary (through plasmids) resistance to antibiotics could be passed through this nanotube network, they manipulated different strains of <i>B.subtilis</i>. We reproduced these experiments and some others related to antibiotic resistance and discussed this matter at length <a href="https://2011.igem.org/Team:Paris_Bettencourt/Atb_exp">here</a>.</p>
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<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>
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<table>
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  <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>
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  </td>
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  <td><b><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/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 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>
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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.
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  <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>
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  <td><em><a href="https://2011.igem.org/Team:Paris_Bettencourt/SinOp">Sin Operon:</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.
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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><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.
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  <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>
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  <td><b><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.
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</table>
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<h2>Others designs we had no time to build</h2>
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<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>
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<h3>Abandonned design for <i>B.subtilis</i></h3>
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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 on the linked page.</li>
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<h3>Designs for <i>B.subtilis-E.coli</i> communication</h3>
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<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>
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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+ 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>
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<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>
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<h4>If communication happens with the cytoplasm</h4>
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<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>
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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: 500px; margin-left: 185px;"></a></br>
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<p><center><b><u>Fig3:</u> Schematics of the communication happening directly to the cytoplasm</b></center></p>
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<p>Here are the designs created in this case.</p>
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<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>
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<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>
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<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>
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<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>
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<p>Finally, encouraged by the results of these antibiotics experiments, the Dubey/Ben-Yehuda team took another round of fluorescent and electronic microscopy pictures, this time involving <i>B.subtilis</i>, <i>E.coli</i> and <i>S.aureus</i>. <em>Nanotubes connected those different species</em>, even though some are Gram-positive (<i>B.subtilis</i> and <i>S.aureus</i>) and the other is Gram-negative (<i>E.coli</i>)!</p>
 
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<h2>The Project</h2>
 
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<p>The existence of the nanotube network discovered by Dubey and Ben-Yehuda is still discussed. We wanted to <em>use synthetic biology to provide new evidence</em> supporting the existence of a new cell-to-cell communication in <i>Bacillus Subtilis</i> and between <i>Bacillus Subtilis</i> and <i>E.coli</i>. Thus, we want to <em>characterize this communication</em> as best as we can using carefully crafted genetic designs. We also aim at <em>proposing new applications</em> combining synthetic biology and the nanotubes network.</p>
 
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<p>Each step of our project corresponds to a new level of understanding of the nanotube network inner mechanisms.</p>
 
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<h4>If communication happens with the periplasm</h4>
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<h3>Preliminary experiments</h3>
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<p>In case the communication happens with the periplasm, we have to think about molecules that can be then transported into the cytoplasm.</p>
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<p>Firstly, we aimed to <em>reproduce</em> the observations made in the initial paper. To this end, we reproduced the <a href="https://2011.igem.org/Team:Paris_Bettencourt/Atb_exp">antibiotic</a> and the <a href="https://2011.igem.org/Team:Paris_Bettencourt/GFP_diff">GFP</a> experiments. We also introduced new hypotheses whenever possible and came up with possible explanations for our results.</p>
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<p>Since we did not have access to an electronic microscopy facility, we were not able to reproduce the most striking pictures of the article. However, we were determined to obtain quantitative and reliable results with synthetic biology approach.</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><u>Fig4:</u> Schematic of the communication happening through the periplasm.</b></center></p>
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<h3>Characterization</h3>
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<p>We had to design more sophisticated approaches. The ideas are the following:</p>
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<p>Our second aim was to <em>characterize</em> the nanotubes: what passes through them and what are the typical diffusion times through the network. We tested if RNA, proteins of different sizes and/or metabolites can pass through and with which ease and rate. The idea is to pass different molecules so that we can characterize the speed and the transfer mechanism. For that purpose, we engineered, using synthetic biology approaches <a href="http://en.wikipedia.org/wiki/BioBrick">[2]</a>, different designs built on this logic:</p>
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<ul><li>An emitter cell that produces a messenger (RNA, protein etc.) </li>
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<li>This messenger passes through the nanotubes and into the receiver cell</li>
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<li>The emitter cell has specific promoters that activates an amplification system</li>
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<li>This amplification system in turn trigger a detection mechanism  we can measure (fluroescence, others)</li></ul>
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<center><img src="https://static.igem.org/mediawiki/2011/e/e6/Schema-presentation.png" style="width:800px"></center>
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<p><b><center><u>Fig4:</u> Global idea for our designs</center></b></p>
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<p>Even though the inter-species (<i>B.subtilis-E.coli</i>) connection seemed more difficult to be reproduced according to the Dubey/Ben-Yehuda paper, we decided to explore it along with the <i>B.subtilis-B.subtilis</i> connection. This was mainly motivated by the overwhelming number of Biobricks available for <i>E.coli</i> when compared to those avalaible for <i>B.subtilis</i>.</p>
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<p>An overview of these steps of the project is available <a href="https://2011.igem.org/Team:Paris_Bettencourt/Designs">here</a>.</p>
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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><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>
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<h3>Proposing a model for transfer mechanism</h3>
 
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<p>The results from the Dubey/Ben-Yehuda paper suggest that there can be an active process that makes the transfer of the cell constituents from the first one to the second faster than the simple diffusion. This hypothesis is to be taken with caution. A completely active process would involve specific transporters whereas the variety of the molecules transported indicate that there are probably no specificity in the transport.</p>
 
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<p>We chose to <em>investigate an alternative mechanism</em>. Based on tension difference between the lipid membrane of two neighbouring cells, we propose a model that could justify the quick transfer through the nanotubes. We call it "assisted diffusion".</p>
 
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<p>The detailed explanation for this assisted diffusion model is available <a href="https://2011.igem.org/Team:Paris_Bettencourt/Modeling/Assisted_diffusion">here</a>.</p>
 
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<div id="citation_box">
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<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>
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<p id="references">References</p>
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<ol>
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<h2>Can we conclude on these designs?</h2>
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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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<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>
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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>
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<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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<p><center><b><u>Fig5:</u> Specification of the master slave design</b></center></p>
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<p>Such a design implies the reversibility of all the sub-systems, the activators and the amplifiers in particular.</p>
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<p>Several potential designs are summed-up below:</p>
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<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>
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<div id="scroll_left"><a href="https://2011.igem.org/Team:Paris_Bettencourt/Project"><img src="https://static.igem.org/mediawiki/2011/0/0a/Arrow-left-big.png" style="width:100%;"></a><a href="https://2011.igem.org/Team:Paris_Bettencourt/Project">Project overview</a></div>
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Revision as of 20:21, 21 September 2011

Team IGEM Paris 2011

Designs overview

Introduction

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.

In the original paper, the author observe directly the molecules that passes through the nanotubes. The idea behind our approach is to use synthetic biology methods to go farther on the sensitivity and the resolution of these experiments. Relying on signal amplification methods and natural and artificial bistable switches to detect at the molecule resolution when a few molecules have diffused from one emittor cell to the receiver.

Main questions behinds

The nanotube discorevy is very recent. Though, lots of question remains unsolved, on the processes of the formation of the tubes, the efficiency of the communication, the extent of the process. The existence itself remains contested by some scientists.

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:

  • How the tubes are forming?
  • What is the speed of the process? (passive or active transport)
  • Can we pass all kind of molecules?
  • Is the communication happens only in between B. subtilis, or can it happens also interspecies or even between E. coli strains?
  • If a communication is established with a Gram positive bacteria, is the communication happening through the periplasm of the cytoplasm?

Specification of our designs

We started designing the project with these the previously stated questions in questions in mind. Using several molecules of different sizes, 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?

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


Fig1: General plan for the experiment design to characterize the nanotubes

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:

  • Use each time the same actuator and the same monitor to have comparable response times in the different systems.
  • 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

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 modeling pages for more details about this assuption.


In B.subtilis, 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:


Fig2: Schematic of the supposed connection between the cells via the nanotubes

Building fast new genetic devices

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.

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..
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.
Sin Operon: 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.
The YFP-TetR/TetO array experiment: 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.
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. A major precaution is to prevent the first cell from sporulating.

Others designs we had no time to build

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 Subtilis/Coli connexions but had no time to do it properly. We nonetheless present all our ideas in this section.

Abandonned design for B.subtilis

  • 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 on the linked page.

Designs for B.subtilis-E.coli communication

The article demonstrates that nanotubes established inside the B.subtilis family can also be formed between B.subtilis and E.coli. 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 B.subtilis. The existence of inter-species nanotubes 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 B.subtilis applied to a Subtilis/Coli connexion, we plan to experiment them in the future since they require very little additional work.

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+ bacteria, like E.coli, 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.

If nanotube communication can be established between E.coli and B.subtilis, it is not known whether the communication happens through the cytoplasm or the periplasm. To test these two hypotheses, two designs were made:

If communication happens with the cytoplasm

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 B.subtilis to E.coli.


Fig3: Schematics of the communication happening directly to the cytoplasm

Here are the designs created in this case.

  • T7 polymerase diffusion: 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 B.subtilis and E.coli.
  • KinA diffusion: 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.
  • 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. A major precaution is to prevent the first cell from sporulating.
  • Amber suppressor tRNA diffusion: 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.

If communication happens with the periplasm

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


Fig4: 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.
  • OmpR diffusion: we need a OmpR- Receptor* E.coli mutant. We produce the OmpR protein in B.subtilis. As long as the OmpR has not diffused from B.subtilis, the signaling cascade cannot be activated. With the rescue by B.subtilis of the OmpR protein, the expression of the reporter gene is activated.

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.

Can we conclude on these designs?

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.