DNA-guided cell-free protein synthesis using a minimal set of purified components has emerged as a versatile platform in constructive biology. The E. coli-based PURE (protein synthesis using recombinant elements) system offers the basic protein synthesis factory in a prospective minimal cell relying on extant molecules. However, there is an urgent need to improve the system's performance and to build a mechanistic computational model that can help interpret and predict gene expression dynamics. Herein, we utilized all three commercially available PURE system variants: PURExpress, PUREfrex and PUREfrex2.0. We monitored apparent kinetics of mRNA and protein synthesis by fluorescence spectroscopy at different concentrations of DNA template. Analysis of polysome distributions by atomic force microscopy, combined with a stochastic model of translation, revealed inefficient usage of ribosomes, consistent with the idea that translation initiation is a limiting step. This preliminary dataset was used to formulate hypotheses regarding possible mechanisms impeding robust gene expression. Next, we challenged these hypotheses by devising targeted experiments aimed to alleviate the current limitations of PUREfrex. We identified depletion of key initiation factors (IFs) by translationally inactive mRNA as a possible inhibitory mechanism. This adverse process could partly be remedied by targeted mRNA degradation, whereas addition of more IFs and of the hrpA RNA helicase had no substantial effects. Moreover, the depletion of tRNAs as peptidyl-tRNAs can become limiting in PUREfrex (but not in PURExpress), which can be alleviated by addition of peptidyl-tRNA-hydrolase (PTH). We attempted to build a new model for PURE system dynamics integrating all experimental observations. Although a satisfying global fit can be obtained in specific conditions (with PTH), a unifying system's level model is still missing.

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.

Replication of DNA-encoded information and its conversion into functional proteins are universal properties of life. In an effort toward the construction of a synthetic minimal cell, we implement here the DNA replication machinery of the Φ29 virus in a cell-free gene expression system. Amplification of a linear DNA template by self-encoded, de novo synthesized Φ29 proteins is demonstrated. Complete information transfer is confirmed as the copied DNA can serve as a functional template for gene expression, which can be seen as an autocatalytic DNA replication cycle. These results show how the central dogma of molecular biology can be reconstituted and form a cycle in vitro. Finally, coupled DNA replication and gene expression is compartmentalized inside phospholipid vesicles providing the chassis for evolving functions in a prospective synthetic cell relying on the extant biology.