Complex tree decision in cells

Natural metabolic regulation is by essence homeostatic and operates through constant variation (usually very small) around an average value, and the gene regulation works the same way. Building an artificial and complex network of metabolic paths requires that some of the decisions and orientations of the recreated metabolism are controlled by the engineer (and not by the cell) and are also transmitted to daughter cells. Recombination based logic gates such as AND, OR, XOR, NOR etc. are perfectly fit to do this kind of user controlled decision tree.

Perfect control of the switch

The cellular level of recombinases is essential. These enzymes can be active enough to switch the gates even with low expression levels, and, as the recombination is not reversible, there should be absolutely no unwanted recombinases expression. The riboregulators of our project were designed to effectively inhibit any uncontrolled expression, particularly in the case of leaky promoter controlling the recombinases gene. This aspect is essential for a calculator (even a bacterial one) were the result should be considered as sure!! However, we also imagined that a quasi-perfect control of the recombinases expression would be essential for any kind of logic gate and that the riboregulator should then be considered as an essential part of the recombinases expression modules.

Universality of recombination based logic gates

The last advantage of the system is its modularity. First, many recombinases can be added to the system, augmenting the possibility of using several gates together. Second, this type of logic gates can be assembled using many output genes. In our example, the output was designed to be visible, but extension of the project envisioned whole metabolic pathways controlled by the gates. Lastly, input genes can also be varied. The riboregulators presented here are designed to incorporate two distinct promoters to perfectly control the recombinases expression, leaving o the user the possibility to introduce any promoter and hence any input signal. The whole system is therefore highly adaptable to the many situations any researcher could envision.

A simple example will convince you (especially this one!!)

A brewer would like to ferment aromatized beer. The pathway for producing menthol was introduced in yeast, but is highly toxic for the cells. Hence, the production should occur only after fermentation (when the beer is at 6% alcohol). Furthermore, two essentials metabolites (A and B) will be used for producing menthol and the yeast needs both for survival.