Team:INSA Toulouse/contenu/project/novel approach



Why design new logic gates?

Artificial regulation networks

Metabolic engineering, one of the branch of Synthetic Biology, consists of producing molecules via biosynthesis, either from assembling existing, natural pathways or via the design of completely artificial metabolic paths. For both natural and artificial pathways, there exist a clear advantage to design genetic logic gates capable of perfectly controlled decisions at the cellular level.

  • The recombination-based genetic logic gates have the capability to permanently switch the state of the gate. Therefore, they are not sensitive to metabolic variations, to retro controlled transcriptional induction or repression, or to changes in metabolic induced changes in enzyme activities.
  • The metabolic switches induced by the doors are strictly controlled even when there are changes in the cell metabolism. This means that our system escapes cell adaptation. In non-recombination based gates that operate with transcriptional regulation, cell metabolic state can modify the output of the gate. In the presented system, it is not possible.
  • The switch is under classical genetic transmission. Contrarily to transcriptional regulation, the phenotype induced by the switch is permanent and is transmitted to all daughter cells. This is also a great manner to genetically amplify the strain bearing the desired switch.

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, increasing 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 to 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. This ensemble of conditions can be assembled into a new (bio)logic module: a triple entry AND gate. The following figure provides a schematic drawing of the problem.

This can be further translated into a biological module. The new triple entry AND gate design would be able to switch three genetic elements. The biological function, here the production of a transcription factor that would control the expression of the menthol synthesis gene, would be achieved only if the three elements are switched in the right direction.

This example demonstrates the power and modularity of recombination-based modules or devices. They are simple to design and construct and hopefully they will constitute new standards for implementing decision trees in cells. In our sense, there is no competition between recombination-based logic gates and other ones. It all depends on the application that would be needed. Recombination-based devices have advantages and drawbacks, and it is up to the user to define the pros and cons of using them in a tailored system.