The bacterial Min protein system was encapsulated in giant unilamellar vesicles (GUVs). Using confocal fluorescence microscopy, we identified several distinct modes of spatiotemporal patterns inside spherical GUVs. For osmotically deflated GUVs, the vesicle shape actively changed in concert with the Min oscillations. The periodic relocation of Min proteins from the vesicle lumen to the membrane and back is accompanied by drastic changes in the mechanical properties of the lipid bilayer. In particular, two types of oscillating membrane‐shape changes are highlighted: 1) GUVs that repeatedly undergo fission into two connected compartments and fusion of these compartments back into a dumbbell shape and 2) GUVs that show periodic budding and subsequent merging of the buds with the mother vesicle, accompanied by an overall shape change of the vesicle reminiscent of a bouncing ball. These findings demonstrate how reaction–diffusion‐based protein self‐organization can directly yield visible mechanical effects on membrane compartments, even up to autonomous division, without the need for coupling to cytoskeletal elements.

Understanding the dynamics of complex enzymatic reactions in highly crowded small volumes is crucial for the development of synthetic minimal cells. Compartmentalised biochemical reactions in cell-sized containers exhibit a degree of randomness due to the small number of molecules involved. However, it is unknown how the physical environment contributes to the stochastic nature of multistep enzymatic processes. Here, we present a robust method to quantify gene expression noise in vitro using droplet microfluidics. We study the changes in stochasticity in cellfree gene expression of two genes compartmentalised within droplets as a function of DNA copy number and macromolecular crowding. We find that decreased diffusion caused by a crowded environment leads to the spontaneous formation of heterogeneous micro-environments of mRNA as local production rates exceed diffusion rates of macromolecules. This heterogeneity leads to a higher probability of the molecular machinery to stay in the same microenvironment, directly increasing the system's stochasticity.Noise is present in all living cells. It has been studied in prokaryotes and eukaryotes 1 , as well as stem 2,3 and cancer cells 4 , and cells expressing viruses 5 . Gene expression is a key example of a complex stochastic enzymatic process. Careful analysis of variations in mRNA and protein levels has revealed the importance of both amplitude and typical decay time of noise and the ability of cells to exploit or suppress noise in gene expression [6][7][8][9] . Unlike deterministic models of gene expression, which are used to predict dynamics over large populations, stochastic models can correctly predict the dynamics of gene expression at the Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use

The formation of cytomimetic protocells that capture the physicochemical aspects of living cells is an important goal in bottom-up synthetic biology. Here, we recreated the crowded cytoplasm in liposome-based protocells and studied the kinetics of cell-free gene expression in these crowded containers. We found that diffusion of key components is affected not only by macromolecular crowding but also by enzymatic activity in the protocell. Surprisingly, size-dependent diffusion in crowded conditions yielded two distinct maxima for protein synthesis, reflecting the differential impact of crowding on transcription and translation. Our experimental data show, for the first time, that macromolecular crowding induces a switch from reaction to diffusion control and that this switch depends on the sizes of the macromolecules involved. These results highlight the need to control the physical environment in the design of synthetic cells.

Secondary structure formation of mRNA, caused by desynchronization of transcription and translation, is known to impact gene expression in vivo. Yet, inactivation of mRNA by secondary structures in cell-free protein expression is frequently overlooked. Transcription and translation rates are often not highly synchronized in cell-free expression systems, leading to a temporal mismatch between the processes and a drop in efficiency of protein production. By devising a cell-free gene expression platform in which transcriptional and translational elongation are successfully performed independently, we determine that sequence-dependent mRNA secondary structures are the main cause of mRNA inactivation in in vitro gene expression.

a b s t r a c tThe cowpea chlorotic mottle virus (CCMV) is a nanoparticle that holds promise for diagnostic and therapeutic applications. The empty virus-like particle, however, is not stable under physiological conditions. Here, we describe a systematic study into the expansion of the assembly properties of a proteinbased block copolymer of the CCMV capsid protein and an elastin-like polypeptide. By systematically changing the hydrophobicity of the stimulus-responsive elastin-like polypeptide block, assembly of the capsid proteins could be achieved at close to physiological conditions. This strategy may prove to be useful in the development of a physiologically stable CCMV capsid variant.