Team:Paris Bettencourt/Experiments/YFP TetR diffusion

From 2011.igem.org

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In the article <a href="http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.2003.03837.x/pdf">[1]</a>, <i>E. coli</i> strains are growing at 20°C to avoid protein agregation but the problem is that nanotube between <i>B. subtilis</i> has been only proved to exist at 37°C.
In the article <a href="http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.2003.03837.x/pdf">[1]</a>, <i>E. coli</i> strains are growing at 20°C to avoid protein agregation but the problem is that nanotube between <i>B. subtilis</i> has been only proved to exist at 37°C.
We test different possibilities : at 37°C or 30°C and concentration of arabinose (0% - 0,1% -0,2%) to deal with protein agregation.
We test different possibilities : at 37°C or 30°C and concentration of arabinose (0% - 0,1% -0,2%) to deal with protein agregation.
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<div style="margin-left:50px; margin-right:50px; padding: 5px; border:2px solid black;"><b><p><em>At 37°C</em>:
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{| border="1" class="wikitable" style="text-align: center;"
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|+tetR:YFP protein fusion : 37°C
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    <li><center><img src="https://static.igem.org/mediawiki/2011/thumb/4/43/FX234_02_YFP01.jpg/800px-FX234_02_YFP01.jpg"><p><u>Fig1:</u> Schematics of the YFP concentration design</center></p> </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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|[[File:FX234_01_Trans01.jpg|450px|thumb|center|tetR:YFP protein fusion inducted with 0,1% arabinose on E. Coli from Dave Lane plasmids.]]
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    <li>GFP can be observed passing through these nanotubes.</li>
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|[[File:FX234_01_YFP01.jpg|450px|thumb|center|tetR:YFP protein fusion inducted with 0,1% arabinose on E. Coli from Dave Lane plasmids.]]
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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>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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|[[File:FX234_02_Trans01.jpg|450px|thumb|center|tetR:YFP protein fusion inducted with 0,2% arabinose on E. Coli from Dave Lane plasmids.]]
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|[[File:FX234_02_YFP01.jpg|450px|thumb|center|tetR:YFP protein fusion inducted with 0,2% arabinose on E. Coli from Dave Lane plasmids.]]
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More pictures and information on the notebook <a href="https://2011.igem.org/Team:Paris_Liliane_Bettencourt/Notebook/2011/08/03/#Kevin">[2]</a>.
More pictures and information on the notebook <a href="https://2011.igem.org/Team:Paris_Liliane_Bettencourt/Notebook/2011/08/03/#Kevin">[2]</a>.

Revision as of 14:26, 12 September 2011

Team IGEM Paris 2011

Experiments of the YFP concentration design

The planning of the experiments is the following : first we have tested the strains from D. Lane containing YFP:tetR and tetO array. Then we constructed/biobricked the YFP:tetR and tetO array system. To finish with the microscopy step and results of this proof of concept between B. subtilis and B. subtilis / E. coli.

Testing the YFP:tetR strains from D. Lane

In the article [1], E. coli strains are growing at 20°C to avoid protein agregation but the problem is that nanotube between B. subtilis has been only proved to exist at 37°C. We test different possibilities : at 37°C or 30°C and concentration of arabinose (0% - 0,1% -0,2%) to deal with protein agregation.

tetR:YFP protein fusion : 37°C
tetR:YFP protein fusion inducted with 0,1% arabinose on E. Coli from Dave Lane plasmids.
tetR:YFP protein fusion inducted with 0,1% arabinose on E. Coli from Dave Lane plasmids.
tetR:YFP protein fusion inducted with 0,2% arabinose on E. Coli from Dave Lane plasmids.
tetR:YFP protein fusion inducted with 0,2% arabinose on E. Coli from Dave Lane plasmids.

More pictures and information on the notebook [2].

Biobricked system construction

Results and microscopy of the proof of concept

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




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