Team:INSA Toulouse/contenu/project/e calculus design
From 2013.igem.org
E. calculus design
General idea
![](https://static.igem.org/mediawiki/2013/6/60/E.caclulusdesign.GeneralIdea.png)
![](https://static.igem.org/mediawiki/2013/0/06/Ecalculus_design.png)
Implementing the whole calculator in one strain
The full DNA construct, general inducer, input system, logic gates, output and carry was clearly too large to fit on a single plasmid (22 kb). Furthermore, we also imagined that switching DNA elements on a multicopy plasmid may not be completely wise, especially when our riboregulators were supposed to exert a strong control over the recombinases expression (i.e. maintaining a very low level of recombinases). Integration of some modules in the E. calculus genome was clearly the solution. We imagined that two different integration sites, one bearing AND1 and AND2, the second bearing XOR1 and XOR2 would be ideal. With this design, recombination events between XOR1 and AND1 or XOR2 and AND2 would lead to chromosomal DNA defects and cell death. The system would then be forced to recombine only the two sites belonging to the same gate.
The general inducer, input and output system as well as the carry can then be assembled on a single plasmid. The general strain would then carry the logic gates and would be stable (no recombinases produced). Upon transformation with the plasmid containing the general inducer, input, output and carry modules, the E. calculus strain would be generated, but still not able to switch before the general inducer put in the medium.
For the final design, a great help was provided by François Cornet and Caroline Schiavon from the LMGM laboratory. They gave us the MG1655 strain that contain special integration sites with two plasmids that would allow these specific integrations.