Team:Paris Bettencourt/GFPLac diffusion

From 2011.igem.org

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Origins of YFP:tetR and tetO Array come from François-Xavier Barre, Andrew Wright and Dave Lane (Kinetics of plasmid segregation, Molecular Microbiology, 2004)</p>
Origins of YFP:tetR and tetO Array come from François-Xavier Barre, Andrew Wright and Dave Lane (Kinetics of plasmid segregation, Molecular Microbiology, 2004)</p>
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<p> In the Ben-Yehuda paper, GFP has been proved to pass though the nanotubes.  We start to build the same experiment but improved by the tetR:YFP - tetO Array system and we used this design as a proof of nanotube concept between<i>B.Subtilis - B.Subtilis</i> and <i>B.Subtilis - E. Coli</i>.</p>
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<p> In the Ben-Yehuda paper, GFP has been proved to pass though the nanotubes.  We start to build the same experiment but improved by the tetR:YFP - tetO Array system and we used this design as a proof of nanotube concept between <i>B.Subtilis - B.Subtilis</i> and <i>B.Subtilis - E. Coli</i>.</p>
<h2>Making the YFP:tetR diffuse through the tube</h2>
<h2>Making the YFP:tetR diffuse through the tube</h2>
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<p><em>In the emittor cell <i>(B. Subtilis)</i></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><em>In the emittor cell <i>(B. Subtilis)</i></em>, we have inserted a expressive system for the YFP:tetR. It contains the promoter pVeg, the RBS for B. Subtilis and the YFP:tetR protein. Production of YFP:tetR will diffuse throught the nanotube to the receiver cell.</p>
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<p><em>In the receiver cell <i>(B. Subtilis or E. Coli)</i></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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<p><em>In the receiver cell <i>(B. Subtilis or E. Coli)</i></em>, there is the tetO array where diffused YFP:tetR will concentrate. The YFP is the monitor of the signal.</p>
<p>The principle of the design is summed up in the image below</p>
<p>The principle of the design is summed up in the image below</p>
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<p>To know more about what we have done on this system and in the experiments, we invite you to visit the correcponding <em>modeling</em> and <em>experiment</em> pages:</p>
<p>To know more about what we have done on this system and in the experiments, we invite you to visit the correcponding <em>modeling</em> and <em>experiment</em> pages:</p>
<ul>
<ul>
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<li><a href="https://2011.igem.org/Team:Paris_Bettencourt/Modeling/T7_diffusion">Modeling</a></li>
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<li><a href="https://2011.igem.org/Team:Paris_Bettencourt/Modeling/YFP_TetR_diffusion">Modeling</a></li>
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<li><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/T7_diffusion">Experiments</a></li>
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<li><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/YFP_TetR_diffusion">Experiments</a></li>
</ul>
</ul>
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Revision as of 18:53, 11 September 2011

Team IGEM Paris 2011

The YFP Concentration design

YFP:tetR is a recombinant fusion protein. It is composed by the Yellow Fluorescent Protein (YFP) and the Tetracycline Repressor Protein (tetR) and bind to the tet operator sequence (tetO). Using the tetO array composed by a 10kb tetO sequence, we can concentrate YFP:tetR in a few loci and increase the fluorescence sensibility. Origins of YFP:tetR and tetO Array come from François-Xavier Barre, Andrew Wright and Dave Lane (Kinetics of plasmid segregation, Molecular Microbiology, 2004)

In the Ben-Yehuda paper, GFP has been proved to pass though the nanotubes. We start to build the same experiment but improved by the tetR:YFP - tetO Array system and we used this design as a proof of nanotube concept between B.Subtilis - B.Subtilis and B.Subtilis - E. Coli.

Making the YFP:tetR diffuse through the tube

In the emittor cell (B. Subtilis), we have inserted a expressive system for the YFP:tetR. It contains the promoter pVeg, the RBS for B. Subtilis and the YFP:tetR protein. Production of YFP:tetR will diffuse throught the nanotube to the receiver cell.

In the receiver cell (B. Subtilis or E. Coli), there is the tetO array where diffused YFP:tetR will concentrate. The YFP is the monitor of the signal.

The principle of the design is summed up in the image below


Fig1: Schematics of the YFP concentration design


Model and experiments

To know more about what we have done on this system and in the experiments, we invite you to visit the correcponding modeling and experiment pages:



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