Cell-free biogenesis of bacterial division proto-rings that can constrict liposomes.

A major challenge towards the realization of an autonomous synthetic cell resides in the encoding of a division machinery in a genetic programme. In the bacterial cell cycle, the assembly of cytoskeletal proteins into a ring defines the division site. At the onset of the formation of the Escherichia coli divisome, a proto-ring consisting of FtsZ and its membrane-recruiting proteins takes place. Here, we show that FtsA-FtsZ ring-like structures driven by cell-free gene expression can be reconstituted on planar membranes and inside liposome compartments. Such cytoskeletal structures are found to constrict the liposome, generating elongated membrane necks and budding vesicles. Additional expression of the FtsZ cross-linker protein ZapA yields more rigid FtsZ bundles that attach to the membrane but fail to produce budding spots or necks in liposomes. These results demonstrate that gene-directed protein synthesis and assembly of membrane-constricting FtsZ-rings can be combined in a liposome-based artificial cell.

De novo synthesized Min proteins drive oscillatory liposome deformation and regulate FtsA-FtsZ cytoskeletal patterns.

The Min biochemical network regulates bacterial cell division and is a prototypical example of self-organizing molecular systems. Cell-free assays relying on purified proteins have shown that MinE and MinD self-organize into surface waves and oscillatory patterns. In the context of developing a synthetic cell from elementary biological modules, harnessing Min oscillations might allow us to implement higher-order cellular functions. To convey hereditary information, the Min system must be encoded in a DNA molecule that can be copied, transcribed, and translated. Here, the MinD and MinE proteins are synthesized de novo from their genes inside liposomes. Dynamic protein patterns and accompanying liposome shape deformation are observed. When integrated with the cytoskeletal proteins FtsA and FtsZ, the synthetic Min system is able to dynamically regulate FtsZ patterns. By enabling genetic control over Min protein self-organization and membrane remodeling, our methodology offers unique opportunities towards directed evolution of bacterial division processes in vitro.