The goal of this research line at the biology/chemistry/biophysics interface is the development of micrometric multicompartimental addressable capsules, which can serve as hosts for biological entities, such as enzymes. The sequestration of individual biomolecules in these robust self-assembled polymer-based host systems, would offer a controlled environment to disentangle complex reaction pathways on introducing selected reactants in a controlled dose or sequence while enzyme activity can be monitored. This can be considered a “bottom-up approach” to Synthetic Biology, harnessing a synthetic minimal cell acting as a functional platform and allowing integration of multiple components in a much simpler and controlled form than natural cells In this paper, we aim at mimicking the complexity of eukaryotic cells that possess a multicompartmentalised structure, via which the cell gains spatiotemporal control over its processes, allowing it to physically separate reaction components and pathways by confining them to different organelles. In order to construct a model system that would allow the study of the effect of compartmentalisation on multistep processes, we have developed a functional and structural cell mimic via a self-assembly approach. A polymersomes-in-polymersome multicompartment system, containing enzyme-filled semi-porous PS-b-PIAT nanoreactors that act as artificial organelles, was prepared to mimic the organisational structure of the cell. A three-enzyme cascade reaction that requires the presence of natural cofactors was used to mimic a natural reaction pathway, where the three different enzymes were confined to both the cytosol and different organelle compartments of the cell mimic. A profluorescent substrate was used to show that the reaction exclusively takes place in the cell-mimics. Furthermore, we demonstrate that the fluorescent reaction product can be locally produced and retained in organelle mimics containing the final enzyme of the cascade reaction. In addition, the use of confocal fluorescence to follow reaction kinetics and to prove that a multicompartmentalized enzymatic reaction is taking place inside single colloidal objects is also in itself a technological challenge of major importance

From compartmentalized polymersomes to biomimetic artificial cell

249th National Meeting (2015)

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