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

Abstract

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.

In vitro synthesis of 32 translation-factor proteins from a single template reveals impaired ribosomal processivity

“…QconCAT purification. QconCAT halves were expressed in BL21(DE3) cells in M9 medium with 15 NH 4 Cl and ampicillin 54 . A pre-culture was diluted 1:100 to a 50-mL expression culture.…”

“…Trypsin digest. Enzymatic digestion of proteins was performed as previously described 54 . Per LC-MS injection, 1.5 µL of PURE system reaction was mixed with 3 µL of 100 mM Tris-HCl pH8.0, 0.3 µL of 20 mM CaCl 2 , and 0.97 µL MilliQ water.…”

Engineering spatiotemporal organization and dynamics in synthetic cells

“…This finding suggests that it may be possible to divide a liposome‐based synthetic cell via “budding” induced by Min oscillations, and without FtsZ. Godino et al (2020) in a separate publication, were also recently successful in cell‐free gene expression of FtsZ‐FtsA ring‐like structures that could constrict liposomes; generating elongated membrane necks and, on occasion, budding vesicles. To date, however, these stepwise additions of divisome components have yet to show consistent abscission of liposomes.…”

“…FtsZ and FtsA have also been shown to form helical co‐polymers that mechanically constrict liposomes and generate narrow necks, but complete division did not occur (Szwedziak, Wang, Bharat, Tsim, & Löwe, 2014). Very recently, FtsA‐FtsZ ring‐like structures were reconstituted by way of a cell‐free gene expression system both on planar membranes and inside liposome compartments (Godino et al, 2019; Godino et al, 2020). The “cytoskeletal structures” were found to constrict the membrane and generate a few budding vesicles.…”

FtsZ Interactions and Biomolecular Condensates as Potential Targets for New Antibiotics

“…The de novo-synthesized MinDE system ( Figure 1 f), mentioned as part of the SLB reconstructions, has been shown to oscillate between membrane and lumen and produce autonomous deformation of liposomes [ 108 ]. The same cell-free gene expression system was employed in liposomes and lipid bilayers to reconstitute FtsZ-FtsA ring-like structures that were able to constrict the liposomes [ 113 ].…”