Team:INSA Toulouse

Transposition of Boolean operators into genetic devices is one of the various goals of Synthetic Biology. There are many difficulties associated with the biological use of Genetic Boolean Operators (GBO) the major being their inherent inability to make a clear difference between the 0 and 1 state, usually because the 0 state is rarely equal to no response in biological systems. Our project will try to tackle this problem, taking advantage of the most recent technology breakthrough in the design of GBO with the use of recombinases as an entry signal to promote the change of state required in the logical gate. A clear advantage of recombinases is that these protein are active at very small concentrations and are therefore very little sensitive to expression variations within a population. Secondly, being a genetic event, recombination will effectively define the two distinct states (0 or 1) required by the logical gate: recombined or not recombined.

We will build one-way logical gates using Serine recombinases, taking advantage of specific recombination sites that do not permit the reversibility of recombination. Therefore, the newly constructed GBO will be transmitted, at the cell level, to all subsequent daughter cells, leading to a perfect amplification of the signal. The general idea of those kinds of genetic logical gates is to use genetics elements (promoters, genes, terminators) placed in different orientations (forward or reverse) between specific, one way only, recombination sites. The input signal is then the expression (or absence of expression) of specific recombinases that will change (or not) the state of the genetic logical gate, leading to any output signal.

As mentioned above, the real difficulty will be to have a perfect (or almost!) 0 state, i.e. no presence of recombinases. To avoid this potential leaky expression, we will implement a strong genetic switch that will prohibit the production of the recombinases even in the (most probable!!!) case of transcriptional background. An elegant way to overcome this problem is to design a riboswitch that avoid translation by binding to the RBS, preventing the leaky transcripts to be translated.

To demonstrate the validity of these two state logic gates, we will create an n-bits full-adder using recombinases-based AND and XOR logical gates. The E. calculus strain should be able to execute full n-bits counting, taking in account the carry at each stage. These systems should approach as much possible the reliability of an electronic two digit device and help the Synthetic Biology community building strong and robust GBOs.