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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 / tetO array : 37°C

tetR:YFP / tetO array inducted with no arabinose on E. Coli from Dave Lane plasmids.	tetR:YFP / tetO array inducted with no arabinose on E. Coli from Dave Lane plasmids.
tetR:YFP / tetO array inducted with 0,2% arabinose on E. Coli from Dave Lane plasmids.	tetR:YFP / tetO array inducted with 0,2% arabinose on E. Coli from Dave Lane plasmids.

tetR:YFP / tetO array : 30°C

tetR:YFP / tetO array inducted with no arabinose on E. Coli from Dave Lane plasmids.	tetR:YFP / tetO array inducted with no arabinose on E. Coli from Dave Lane plasmids.
tetR:YFP / tetO array inducted with 0,2% arabinose on E. Coli from Dave Lane plasmids.	tetR:YFP / tetO array inducted with 0,2% arabinose on E. Coli from Dave Lane plasmids.

More pictures and information on the notebook [2].

Biobricked system construction

YFP:tetR construction

Fig1: Cloning plan of YFP:tetR construction

TetO array construction

Fig2: Cloning plan of TetO array 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