A minimal gene expression machinery has been encapsulated within large lipid vesicles that are immobilized on a microscope coverslip for fluorescence imaging (see picture). The engineered vesicle membrane acts as an exchange platform that enables the uptake of all necessary nutrients and tRNAs supplied in the outside environment, which initiates the internal synthesis of green fluorescent proteins (GFP) from a DNA template. Red: membrane dye, green: synthesized GFP.

Reconstituting an elementary gene expression system inside self-assembled lipid vesicles to mimic the cellular synthesis machinery is at the core of the development of a minimal cell following a bottom-up synthetic biology approach. The ability to operate the expression of multiple genes in a controlled manner and to generate the output proteins with predictable dynamics in liposomes relies on the link between genotype and phenotype. Here, we established this link in surface-tethered liposomes producing proteins from a linear DNA template using a reconstituted transcription/translation/aminoacylation apparatus fuelled by external supply of feedstock. The amounts of entrapped DNA molecules and synthesized proteins were visualized by fluorescence confocal microscopy in individual vesicles. We showed that there exists no linear correlation between the amount of encapsulated genes and the level of output proteins, which is a consequence of the compositional heterogeneity between liposomes due to the low-copy number of some constituents, as well as interfacing differences with the nutrient-containing environment. In order to decouple gene activity from those sources of variability and, thus, infer the probabilistic occupancy of transcriptionally active genes in protein synthesizing liposomes, we developed a dual gene expression assay consisting of the production of two fluorescent reporter proteins of distinguishable colors from two different DNA templates. The stochastic color-coding of the vesicles was analyzed and compared to the color pattern expected from a Poisson distribution of encapsulated genes. Unexpectedly, we found that the apparent number of transcriptionally active DNA molecules in liposomes corresponds only to ca. 10% of the bulk concentration. We believe that our study provides new insights about the relationship between the genotype and phenotype in protein synthesizing liposomes, which is of primary importance toward the construction of a programmable artificial cell implemented with regulatory gene networks of predictable dynamics.

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The compartmentalization of a cell-free gene expression system inside a self-assembled lipid vesicle is envisioned as the simplest chassis for the construction of a minimal cell. Although crucial for its realization, quantitative understanding of the dynamics of gene expression in bulk and liposome-confined reactions is scarce. Here, we used two orthogonal fluorescence labeling tools to report the amounts of mRNA and protein produced in a reconstituted biosynthesis system, simultaneously and in real-time. The Spinach RNA aptamer and its fluorogenic probe were used for mRNA detection. Applying this dual-reporter assay to the analysis of transcript and protein production inside lipid vesicles revealed that their levels are uncorrelated, most probably a consequence of the low copy-number of some components in liposome-confined reactions. We believe that the stochastic nature of gene expression should be appreciated as a design principle for the assembly of a minimal cell.

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The construction of an irreducible minimal cell having all essential attributes of a living system is one of the biggest challenges facing synthetic biology. One ubiquitous task accomplished by any living systems is the division of the cell envelope. Hence, the assembly of an elementary, albeit sufficient, molecular machinery that supports compartment division, is a crucial step towards the realization of self-reproducing artificial cells. Looking backward to the molecular nature of possible ancestral, supposedly more rudimentary, cell division systems may help to identify a minimal divisome. In light of a possible evolutionary pathway of division mechanisms from simple lipid vesicles toward modern life, we define two approaches for recapitulating division in primitive cells: the membrane deforming protein route and the lipid biosynthesis route. Having identified possible proteins and working mechanisms participating in membrane shape alteration, we then discuss how they could be integrated into the construction framework of a programmable minimal cell relying on gene expression inside liposomes. The protein synthesis using recombinant elements (PURE) system, a reconstituted minimal gene expression system, is conceivably the most versatile synthesis platform. As a first step towards the de novo synthesis of a divisome, we showed that the N-BAR domain protein produced from its gene could assemble onto the outer surface of liposomes and sculpt the membrane into tubular structures. We finally discuss the remaining challenges for building up a self-reproducing minimal cell, in particular the coupling of the division machinery with volume expansion and genome replication.

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