Team:Paris Bettencourt/GFPLac diffusion

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=The YFP Concentration design=
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<h1>The YFP Concentrator design</h1>
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YFP:tetR is a recombinant fusion protein composed by 1357 bp
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<h2>Introduction</h2>
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Their origin come from François-Xavier Barre, Andrew Wright and Dave Lane (
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== Making the YFP:tetR diffuse through the tube ==
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<p>One of the main result of Dubey and Ben-Yehuda papers proving the existence of nanotubes is the evidence of GFP diffusion from  B.subtilis cells one to another.
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However the resulting fluorescence from this diffusion is quite weak supposedly due to the limited number of molecules passing through nanotubes.
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One alternative to get a better fluorescence signal with the same amount of fluorescent molecules is to concentrate them in foci. In order to do this we decided to use an already existing system based on a <em>YFP:TetR</em> fusion protein.
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<p> YFP:TetR is composed of Yellow Fluorescent Protein (YFP) and the Tetracycline Repressor Protein (TetR) that binds to the Tet operator sequence (TetO). Using the TetO array composed of a 10kb repeat of TetO sequences, we can concentrate YFP:TetR in several loci and increase the fluorescence sensitivity.
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The two different constructs, 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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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.<br>
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<p> We use this design as a proof of the nanotube concept between <i>B.Subtilis - B.Subtilis</i> and <i>B.Subtilis - E. Coli</i>.</p>
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[[span style="color:blue"]]In the emittor cell[[/span]], we have to over express the T7 polymerase for them to have a chance to pass through the tube. As we said in the general overview 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.<br>
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<h2>Making the YFP:tetR diffuse through the tube</h2>
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[[span style="color:blue"]]In the receiver cell[[/span]], 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.
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<p><em>In the emitter cell <i>(B. Subtilis)</i></em>, we have inserted an expression system for the YFP:tetR. It contains the constitutive promoter pVeg, the RBS for <i>B.subtilis</i> and the YFP:tetR protein. Constitutively expressed YFP:tetR molecules will diffuse through the nanotube to the receiver cell.</p>
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The principle of the design is summed up in the image below
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[[File:yfptetrdesign2.jpg|703px|thumb|center|the YFP:tetR design]]
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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>
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== Model and experiments ==
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<p>The principle of the design is summed up in the image below</p>
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<br/>
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<center><img src="https://static.igem.org/mediawiki/2011/5/56/TetR-YFP4.jpg">
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<p><u>Fig1:</u> Schematics of the YFP concentration design</center></p>
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<br/>
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<h2>Model and experiments</h2>
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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 corresponding <em>diffusion modeling</em> and <em>experiment</em> pages:</p>
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<ul>
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<li><em><a href="https://2011.igem.org/Team:Paris_Bettencourt/Modeling/Diffusion">Diffusion modelling</a></em></li>
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<li><em><a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/YFP_TetR_diffusion">Experiments</a></em></li>
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</ul>
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<br/>
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Latest revision as of 02:23, 22 September 2011

Team IGEM Paris 2011

The YFP Concentrator design

Introduction

One of the main result of Dubey and Ben-Yehuda papers proving the existence of nanotubes is the evidence of GFP diffusion from B.subtilis cells one to another. However the resulting fluorescence from this diffusion is quite weak supposedly due to the limited number of molecules passing through nanotubes. One alternative to get a better fluorescence signal with the same amount of fluorescent molecules is to concentrate them in foci. In order to do this we decided to use an already existing system based on a YFP:TetR fusion protein.

YFP:TetR is composed of Yellow Fluorescent Protein (YFP) and the Tetracycline Repressor Protein (TetR) that binds to the Tet operator sequence (TetO). Using the TetO array composed of a 10kb repeat of TetO sequences, we can concentrate YFP:TetR in several loci and increase the fluorescence sensitivity. The two different constructs, YFP:TetR and TetO Array, come from François-Xavier Barre, Andrew Wright and Dave Lane (Kinetics of plasmid segregation, Molecular Microbiology, 2004)

We use this design as a proof of the nanotube concept between B.Subtilis - B.Subtilis and B.Subtilis - E. Coli.

Making the YFP:tetR diffuse through the tube

In the emitter cell (B. Subtilis), we have inserted an expression system for the YFP:tetR. It contains the constitutive promoter pVeg, the RBS for B.subtilis and the YFP:tetR protein. Constitutively expressed YFP:tetR molecules will diffuse through 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 corresponding diffusion modeling and experiment pages:


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