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Team:Paris Bettencourt/Experiments/YFP TetR diffusion
From 2011.igem.org
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.At 37°C:
Fig1: Schematics of the YFP concentration design
- A nanotube network can be observed through electronic microscopy between B.subtilis cells.
- GFP can be observed passing through these nanotubes.
- Antibiotic resistance can be transfered between B.subtilis cells or between B.subtilis and E.coli, both in a hereditary and a non-hereditary way.
- Nanotubes connecting different species (B.subtilis, E.coli and S.aureus) have been oberved with electronic microscopy.
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
