Biological Modules

Riboregulation System

In order to better control the expression of the recombinases, we design a riboregulation system to have well controlled promoters before our recombinases.

How does it work ?

This is the basic system : Two inductible promoters each followed by a RNA sequence

The blue and the red regions are regions which code for little RNA sequences.


When the promoter P2 is activated and not the P1, the red sequence will be transcripted. This red sequence possesses a similarity with the rbs sequence (in red). So when transcripted the red sequence will confine the rbs and prevent the recombinase from being translated.

When both promoters are activated, both sequences (the blue and the red one) are transcripted. The blue RNA sequence possesses a stronger similiraty with the red one. When transcripted the blue and the red sequences will match together and release the rbs to permit the translation of the recombinase.

Our strategy

These publications "Genetic switchboard for synthetic biology applications" and "Engineered riboregulators enable post-transcriptional control of gene expression"inspired us. In their work they present one riboregulation system with this idea, we choose to design other riboswitches keeping some sequences from the original one and changing some nucleotides. It will aim use to have one riobregulation system specific for each input, then to do not have conflict. Furthemore, it will give use the possibility to characterize and compare the systems.

Deeper in the design: RNA secondary structures formation

Here are presented secondary structures of two riboregulators, R0 and R1. The RBS blocking RNA segregates the RBS with a strong hairspin. The interfering RNA has a free-end sequence complementary to an unlocking sequence of the RBS blocking RNA. When they are both transcripted, the interaction between the two RNAs allows the RBS sequence to be free and the translation to occur.

R0 riboregulator

R0 RBS blocking RNA
The free energy of the thermodynamic ensemble is -10.14 kcal/mol.

R0 interfering RNA
The free energy of the thermodynamic ensemble is -23.27 kcal/mol.

R1 riboregulator

R1 RBS blocking RNA
The free energy of the thermodynamic ensemble is -8.72 kcal/mol.

R1 interfering RNA
The free energy of the thermodynamic ensemble is -24.10 kcal/mol.

Simulation done on RNAfold Webserver.

References :
Gruber AR, Lorenz R, Bernhart SH, Neuböck R, Hofacker IL.
The Vienna RNA Websuite. Nucleic Acids Res. 2008.

RNA parameters are described in :
Mathews DH, Disney MD, Childs JL, Schroeder SJ, Zuker M, Turner DH. (2004) Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure. Proc Natl Acad Sci U S A 101(19):7287-92.