FXL-T1

Implementation of post-transcriptional regulated cell-free circuits into microfluidic chemostat

Description

With the emergence of synthetic biology, cell-free transcription-translation (TX-TL) systems have become powerful tools to study complex biochemical circuits in vitro. They have turned out to be a platform of choice for approaching such networks in a bottom-up perspective. By expressing the genes of interests outside of a highly complex cellular environment, we have access to the development of quantitative models lightened of most of the characterized noise of the living systems.

Objective

In our project, we will use a system based on the TX-TL machineries of E. coli to build gene circuits controlled at different levels of expression. Modulations of sigma factors along with the endogenous core polymerase have demonstrated their efficiency to build elementary circuit motifs such as several stages cascades, AND gate or negative feedback loop. [1]

Firstly, we propose to expand this toolbox of transcription-regulated parts by including small non-coding mRNA elements, known as riboswitches, which bind to small ligands and are able to tune the production of the self-encoded protein depending on their binding state with the effector molecule [2]. They would allow a supplementary level of control on the post-transcriptional events. More complex circuits based either on combination or sequence of gates will be envisaged.

Depending on their background and interests, the master student could work either on the modeling part or on the experimental side. Riboswitches On/Off states controlled by the ligand concentration over time is therefore a convenient way for going towards highly efficient and versatile synthetic biological circuits. One advantage of cell-free systems is that it supposes to offer an easy access to the reaction (open system). Though in practice, the batch reaction format prevents any easy modification of the TX-TL mix in an automatic and precise way. This drawback leads directly to another one: As we cannot easily dilute the mix with new nutrients or fresh enzymes, the system is going to saturation in a few hours and complex feedback loops are generally impossible to obtain.

Therefore, the second main axis of this project will be to design and test microfluidics nano-reactors, which offer several advantages over the traditional batch mode. By bringing new reagents and ligands to the reaction mix at precise intervals of time, we could finally implement highly controllable networks under steady-state condition. [3]

Prerequisites

Depending on their background and interests, the master student could work either on the modeling part or on the experimental side.

Literature

[1] Shin, J., Noireaux, V. An E. coli cell-free expression toolbox: application to synthetic gene circuits and synthetic cell. ACS Synthetic Biology 1(1), 29-41 (2012)

[2] Groher F, Suess B. Synthetic riboswitches – A tool comes of age. Biochim Biophys Acta pii: S1874-9399(14)00111-4. doi: 10.1016/j.bbagrm.2014.05.005.

[3] Implementation of cell-free biological networks at steady-state. Niederholtmeyer H., Stepanova V., and Maerkl S.J. PNAS 110(40):15985-15990. DOI: 10.1073/pnas.1311166110

Contact

Francois-Xavier Lehr