Team:Paris Bettencourt/T7 diffusion

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<h1>The T7 RNA polymerase design</h1>
<h1>The T7 RNA polymerase design</h1>
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<p>Bacteriophage T7 RNA polymerase is a DNA-dependent RNA polymerase from the T7 bacteriophage genome.  The enzyme is composed of a single polypeptide chain of 880 amino acids. It catalyzes the processive polymerization of messenger RNA from nucleoside triphosphate precursors by using one strand of DNA as a template .  This enzyme is known to have a stringent specificity for its promoter, that is orthogonal to the other promoterr of the cell.</p>
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<p><em>Bacteriophage T7 RNA polymerase</em> is a DNA-dependant RNA polymerase from the T7 bacteriophage genome.  The enzyme is composed of a single polypeptide chain of 880 amino acids. It catalyzes the processive polymerization of messenger RNA from nucleoside triphosphate precursors by using one strand of DNA as a template .  This enzyme is known to have a stringent specificity for its promoter, that is orthogonal to the other promoters of the cell.</p>
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<p>In our designs, we wanted a protein to pass through the tube and trigger a signal in the receiver cell. We see here that T7 RNA polymerase si a very good candidate for sugh system. That's why we used as the biggest<a href="https://2011.igem.org/Team:Paris_Bettencourt/Designs"> of our proof of principle molecules.</a></p>
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<p>In our designs, we wanted a protein to pass through the tubes and trigger a signal in the receiver cell. T7 RNA polymerase is considered as a very good candidate for such systems. This explains why it is used as the biggest<a href="https://2011.igem.org/Team:Paris_Bettencourt/Designs"> of our proof of principle molecules.</a></p>
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<img src="http://upload.wikimedia.org/wikipedia/commons/e/e3/T7_RNA_polymerase.jpg" width=200px><br/>
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<b><u>Fig1:</u></b> Cristallographic structure of RNA polymerase. <a ref="http://en.wikipedia.org/wiki/T7_RNA_polymerase">(source)</a></center>
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<h2>Making the T7 RNA polymerase diffuse through the tube</h2>
 
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<p>Calculating the radius of the RNA polymerase we noticed that it is smaller than the average size of the nanotubes we can measure from the Ben-Yehuda paper. So we made the assumprtion that such a big molecule has a chance to pass through the tubes, and we start building the design.</p>
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<h2>Making the T7 RNA polymerase diffuse through the tubes</h2>
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<p><em>In the emittor cell</em>, we have to over express the T7 polymerase for them to have a chance to pass through the tube. As we said in the <a href="https://2011.igem.org/Team:Paris_Bettencourt/Designs">general overview</a> the production of T7 polymsease is over the control of an IPTG inducible promoter design to have a slow response by the over-expression of LacI in the cell. The RFP, placed on the same mRNA, is behaving like a reporter of the quantity of the produced T7 polymerase.</p>
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<p>When we calculated the radius of the T7 RNA polymerase we noticed that it is smaller than the average size of the nanotubes we can measure from the Dubey and Ben-Yehuda paper. We made the hypothesis that such a big molecule has the chance to pass through the tubes and we started building the design to test it.</p>
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<p><em>In the receiver cell</em>, a system, sensitive to the T7 polymerase will be activated if one T7 polymerase reach on of its promoter, present in a few plasmids of the receiver cell (low copy). The system is self amplifying and the GFP is produced as a monitor of the signal.</p>
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<li><em>In the emitter cell</em>, we have to over express the T7 polymerase. As we said in the <a href="https://2011.igem.org/Team:Paris_Bettencourt/Designs">general overview</a> the production of T7 polymerase is under the control of an IPTG inducible promoter design. The RFP, placed on the same mRNA is a reporter of the quantity of the produced T7 polymerase.</li>
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<p>The principle of the design is summed up in the image below</p>
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<li><em>In the receiver cell</em>, a system sensitive to the T7 polymerase will be activated if a T7 polymerase reaches its promoter, present in a few plasmids of the receiver cell (low copy plasmids). The system is self-amplifying and the GFP is produced as a monitor of the signal.</li>
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<img src="https://static.igem.org/mediawiki/2011/a/a5/T7_diffusion_principle.jpg">
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<p>The principles of the design are summed up in the image below:</p>
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<p><center><u>Fig1:</u> Schematics of the T7 polymerase design</center></p>  
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<h2>T7 polymerase as a good quality auto-amplifier</h2>
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<img src="https://static.igem.org/mediawiki/2011/9/99/T7_diffusion.jpg" width=800px/>
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</p><b><u>Fig2:</u></b> Schematic summary of the T7 diffusion device</p>
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<p>One of our firt concert was the potential leakage from the auto amplifier. We are in biological systems, that is to say we a noisy system. The promoter is known to be very orthogonal from the one of the endogenic polymerase. However, we had to deal with several problem. We invite you to see the experiments page for data about these problems we faced.</p>
 
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<p>As we where not working in a synthetic biology plasmid (we designed our own <a href="https://2011.igem.org/Team:Paris_Bettencourt/MultiHost">multy host vector</a>), designed to be silent, we had to add several stop before the promoters.</p>
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<h2>T7 polymerase as an auto-amplifier</h2>
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<p>One of our first concerns was the potential leaking from the auto-amplifier. Biological systems tend to be noisy. The promoter is known to be very orthogonal to the metabolism of the host. However, we had to deal with several problems. We invite you to see the experiments page for data about this.</p>
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<p>We could not work in a synthetic biology plasmid since it does not exist for <i>B.subtilis</i>. We had to create and use a home made, non biobricked multihost plasmid, not designed to be silent. We had to add several terminators before the pT7 promoters in order to prevent the leakage.</p>
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<p>As an alternative solution, we also constructed a GFP with a T7 promoter. This system is supposed to have a lower response, but without auto-amplification, leaking should be reduced to a minimum.</p>
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<h2>Model and experiments</h2>
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<p>To know more about what we have done with this system, we invite you to visit the corresponding <em>modeling</em> and <em>experiment</em> pages:</p>
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<li><em><a href="https://2011.igem.org/Team:Paris_Bettencourt/Modeling/T7_diffusion">Modeling</a></em></li>
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<li><em><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/T7_diffusion">Experiments</a></em></li>
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<div id="scroll_right"><a href="https://2011.igem.org/Team:Paris_Bettencourt/Modeling/T7_diffusion"><img src="https://static.igem.org/mediawiki/2011/e/e0/Arrow-right-big.png" style="width:100%;"></a><a href="https://2011.igem.org/Team:Paris_Bettencourt/Modeling/T7_diffusion">Model for T7</a></div>
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Latest revision as of 01:40, 22 September 2011

Team IGEM Paris 2011

The T7 RNA polymerase design

Bacteriophage T7 RNA polymerase is a DNA-dependant RNA polymerase from the T7 bacteriophage genome. The enzyme is composed of a single polypeptide chain of 880 amino acids. It catalyzes the processive polymerization of messenger RNA from nucleoside triphosphate precursors by using one strand of DNA as a template . This enzyme is known to have a stringent specificity for its promoter, that is orthogonal to the other promoters of the cell.

In our designs, we wanted a protein to pass through the tubes and trigger a signal in the receiver cell. T7 RNA polymerase is considered as a very good candidate for such systems. This explains why it is used as the biggest of our proof of principle molecules.

            

Fig1: Cristallographic structure of RNA polymerase. (source)

Making the T7 RNA polymerase diffuse through the tubes

When we calculated the radius of the T7 RNA polymerase we noticed that it is smaller than the average size of the nanotubes we can measure from the Dubey and Ben-Yehuda paper. We made the hypothesis that such a big molecule has the chance to pass through the tubes and we started building the design to test it.

  • In the emitter cell, we have to over express the T7 polymerase. As we said in the general overview the production of T7 polymerase is under the control of an IPTG inducible promoter design. The RFP, placed on the same mRNA is a reporter of the quantity of the produced T7 polymerase.
  • In the receiver cell, a system sensitive to the T7 polymerase will be activated if a T7 polymerase reaches its promoter, present in a few plasmids of the receiver cell (low copy plasmids). The system is self-amplifying and the GFP is produced as a monitor of the signal.

The principles of the design are summed up in the image below:


Fig2: Schematic summary of the T7 diffusion device

T7 polymerase as an auto-amplifier

One of our first concerns was the potential leaking from the auto-amplifier. Biological systems tend to be noisy. The promoter is known to be very orthogonal to the metabolism of the host. However, we had to deal with several problems. We invite you to see the experiments page for data about this.

We could not work in a synthetic biology plasmid since it does not exist for B.subtilis. We had to create and use a home made, non biobricked multihost plasmid, not designed to be silent. We had to add several terminators before the pT7 promoters in order to prevent the leakage.

As an alternative solution, we also constructed a GFP with a T7 promoter. This system is supposed to have a lower response, but without auto-amplification, leaking should be reduced to a minimum.

Model and experiments

To know more about what we have done with this system, we invite you to visit the corresponding modeling and experiment pages:



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