The biosynthesis of proteins from genomic DNA is a universal process in every living organism. Building a synthetic cell using separate biological parts hence implies to reconstitute a minimal gene expression apparatus and to compartmentalize it in a cell-mimicking environment. Previous studies have demonstrated that the PURE (Protein synthesis Using Recombinant Elements) system could be functionally encapsulated inside lipid vesicles. However, quantitative insights on functional consequences of spatial confinement of PURE system reactions remain scarce, which has hampered the full exploitation of gene-expressing liposomes as the fundamental unit to build an artificial cell. We report on direct imaging of tens of thousands of gene-expressing liposomes per sample allowing us to assess sub-population features in a statistically relevant manner. Both the vesicle size (diameter  <10 μm) and lipid composition (mixture of phospholipids with zwitterionic and negatively charged headgroups, including cardiolipin) are compatible with the properties of bacterial cells. Therefore, our liposomes provide a suitable chassis to host the Escherichia coli-derived PURE translation machinery and other bacterial processes in future developments. The potential of high-content imaging to identify rare phenotypes is demonstrated by the fact that a subset of the liposome population exhibits a remarkably high yield of synthesized protein or a prolonged expression lifespan that surpasses the performance of ensemble liposome-averaged and bulk reactions. Among the three commercial PURE systems tested, PUREfrex2.0 offers the most favorable phenotypes displaying both high yield and long protein synthesis lifespan. Moreover, probing membrane permeability reveals a large heterogeneity amongst liposomes. In situ expression and membrane embedding of the pore-forming connexin leads to a characteristic permeability time profile, while increasing the fraction of permeable liposomes in the population. We see diversity in gene expression dynamics and membrane permeability as an opportunity to complement a rational design approach aiming at further implementing biological functions in liposome-based synthetic cells.

Original languageEnglish
Article number045002
Number of pages14
JournalPhysical Biology
Issue number4
Publication statusPublished - 2019

    Research areas

  • synthetic cell, minimal cell, PURE system, cell-free gene expression, liposome

ID: 53623083