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Team:Paris Bettencourt/GFPLac diffusion
From 2011.igem.org
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<h1>The YFP Concentration design</h1> | <h1>The YFP Concentration design</h1> | ||
| - | <p><em>YFP:tetR</em> is a recombinant fusion protein. It is composed by | + | <p><em>YFP:tetR</em> is a recombinant fusion protein. It is composed by the Yellow Fluorescent Protein (YFP) and the Tetracycline Repressor Protein (tetR) and bind to the tet operator sequence (tetO). Using the tetO array composed by a 10kb tetO sequence, we can concentrate YFP:tetR in a few loci and increase the fluorescence sensibility. |
| - | + | Origins of YFP:tetR and tetO Array come from François-Xavier Barre, Andrew Wright and Dave Lane (Kinetics of plasmid segregation, Molecular Microbiology, 2004)</p> | |
<p> In the Ben-Yehuda paper, GFP has been proved to pass though the nanotubes. We start to build the same experiment but improved by the tetR:YFP - tetO Array system and we used this design as a proof of nanotube concept between<i>B.Subtilis - B.Subtilis</i> and <i>B.Subtilis - E. Coli</i>.</p> | <p> In the Ben-Yehuda paper, GFP has been proved to pass though the nanotubes. We start to build the same experiment but improved by the tetR:YFP - tetO Array system and we used this design as a proof of nanotube concept between<i>B.Subtilis - B.Subtilis</i> and <i>B.Subtilis - E. Coli</i>.</p> | ||
Revision as of 18:41, 11 September 2011
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) and bind to the tet operator sequence (tetO). Using the tetO array composed by a 10kb tetO sequence, we can concentrate YFP:tetR in a few loci and increase the fluorescence sensibility. Origins 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 start to build the same experiment but improved by the tetR:YFP - tetO Array system and we used this design as a proof of nanotube concept betweenB.Subtilis - B.Subtilis and B.Subtilis - E. Coli.
Making the YFP:tetR diffuse through the tube
In the emittor cell (B. Subtilis), we have to over express the T7 polymerase for them to have a chance to pass through the tube. As we said in the general overview the production of T7 polymsease is over the control of an IPTG inducible promoter design to have a slow response by the over-expression of LacI in the cell. The RFP, placed on the same mRNA, is behaving like a reporter of the quantity of the produced T7 polymerase.
In the receiver cell (B. Subtilis or E. Coli), a system, sensitive to the T7 polymerase will be activated if one T7 polymerase reach on of its promoter, present in a few plasmids of the receiver cell (low copy). The system is self amplifying and the GFP is produced as a 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 correcponding modeling and experiment pages:
