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Team:Paris Bettencourt/GFPLac diffusion
From 2011.igem.org
The YFP Concentrator design
Introduction
One of the main result of Dubey and Ben-Yehuda papers proving the existence of nanotubes is the existence of GFP diffusion from B.subtilis cells one to another. However the resulting fluorescence from this diffusion is quiet weak supposedly due to the limited number of molecules passing through the nanotube. 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 sequence, we can concentrate YFP:TetR in several loci and increase the fluorescence sensibility. 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)
In the Dubey and Ben-Yehuda paper, GFP has been proved to pass though the nanotubes but the number of GFP molecules is not enough to get a clear fluorescence signal. We will recreate the same experiment but improve it with the TetR:YFP - TetO Array system. 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:
