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Team:Paris Bettencourt/GFPLac diffusion
From 2011.igem.org
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) that binds to the tet operator sequence (tetO). Using the tetO array composed of a 10kb tetO sequence, we can concentrate YFP:tetR in several loci and increase the fluorescence sensibility. The strains 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 will recreate the same experiment but improved it with the tetR:YFP - tetO Array system. We used 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 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 corresponding diffusion modeling and experiment pages:
