Hybrid COmpartments as SYnthetic Cells – COSYCELL

Producing synthetic (artificial) cells is of interest for an industrial point of view for the directed synthesis of chemicals (bioreactors) but also for understanding the origin of life. The ‘top-down’ approach principally developed at the J. Craig Venter Institute aims at minimizing bacterial genome, studying the growing ability of mutants deleted for several genes to identify the set of components vital for life of minimal systems. The other fruitful and complementary line of research has involved a ‘bottom-up’ approach aiming at producing artificial compartments drawing on a combination of natural biomolecules and synthons to mimic cells by incorporating i) cellular extracts containing proteins and nucleic acids required for DNA replication and ii) enzymes in order to develop biochemical reactions and reactors. To date, numerous studies have focused on compartments based on either vesicles or water in water emulsions. However, up to now, the production of synthetic cells has not yet been achieved.
The main reason is that vesicles used to mimic cells, which bear a lipid bilayer, do not spontaneously encapsulate chemicals. In contrast, water in water emulsions (including coacervates) do spontaneously sequester chemicals but lack membrane.
CosyCell aims at developing hybrid systems made of water in water emulsions/vesicles in order to gather the advantages of both models in only one. These hybrid compartments will be able to spontaneously sequester chemicals and will bear a membrane to control exchange of solutes with the outer medium. The potential of these hybrid compartments to the production of bioreactors and synthetic cells will be finally studied.

We will be using 2 kinds of systems i) a neutral composed by PEG and dextran mixtures and ii) complex coacervates made of positively and negatively charged polkymers or polyions. 3 methods will be used to cover the droplets with a lipid membrane. The first is to use fatty acids, the polymorphism of which depends on the pH (or light when using azobenzene derivatives) to perform a micelle to vesicle transition. The second will use phospholipids, which will be deposited at the surface of droplets by conventional methods (lipid supported bilayers). The last one involve bola-lipids that bear 2 different polar heads to form a monolayer at the surface of droplets.

Our hybrid systems will bear a lipid membrane that could protect and prevent release of encapsulated chemicals. However, these compartments need to be fed by solutes to be used as bioreactors or synthetic cells. Then, we will address the question of membrane permeability by incorporating pore-forming proteins and/or transporters within the membrane, creating hybrid proteo-compartments.
Finally, our hybrid proteo-compartments being capable of encapsulating proteins, bacteria and/or transcription-translation (TX-TL) systems, these will be used as bioreactors and synthetic cells. Encapsulating enzymes in hybrid compartments that can be fed by their substrate via transporters or pore-forming proteins will yield bioreactors, the activity of which could be easily followed by fluorescence techniques. Encapsulation of bacterial extract or TX-TL systems will allow synthesis of fluorescent proteins within hybrid compartments, a ‘life-like’ experiment as a step to the production of synthetic cells.