"
Team:Paris Bettencourt/T7 diffusion
From 2011.igem.org
The T7 RNA polymerase design
Bacteriophage T7 RNA polymerase is a DNA-dependent RNA polymerase from the T7 bacteriophage genome. The enzyme is composed of a single polypeptide chain of 880 amino acids. It catalyzes the processive polymerization of messenger RNA from nucleoside triphosphate precursors by using one strand of DNA as a template . This enzyme is known to have a stringent specificity for its promoter, that is orthogonal to the other promoterr of the cell.
In our designs, we wanted a protein to pass through the tube and trigger a signal in the receiver cell. We see here that T7 RNA polymerase si a very good candidate for sugh system. That's why we used as the biggest of our proof of principle molecules.
Making the T7 RNA polymerase diffuse through the tube
Calculating the radius of the RNA polymerase we noticed that it is smaller than the average size of the nanotubes we can measure from the Ben-Yehuda paper. So we made the assumprtion that such a big molecule has a chance to pass through the tubes, and we start building the design.
In the emittor cell, 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, 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
T7 polymerase as a good quality auto-amplifier
One of our firt concert was the potential leakage from the auto amplifier. We are in biological systems, that is to say we a noisy system. The promoter is known to be very orthogonal from the one of the endogenic polymerase. However, we had to deal with several problem. We invite you to see the experiments page for data about these problems we faced.
As we where not working in a synthetic biology plasmid (we designed our own multy host vector), designed to be silent, we had to add several stop before the promoters.
