Cells, the building blocks of life, are vastly complex. This complexity confers to every living organism the ability to maintain oneself, reproduce oneself, and evolve. Creating a minimal system from nonliving components that is capable of self-maintenance, self-reproduction, and evolvability, will greatly increase our understanding of life. Essential features of every cell, synthetic or otherwise, are the compartment, a form of information transfer, and the ability to proliferate. In chapter 1, I argue that autonomy, that is self-governance, is another key characteristic of life. Therefore, to create a synthetic cell, aforementioned features should be recapitulated in an autonomous manner. This informs the synthetic cell design that is adhered to in this thesis. Phospholipid vesicles, so called liposomes, serve as a compartment. Reconstitution of the central dogma of molecular biology by cell-free gene expression with the PURE system enables the use of a genetic program encoded on DNA. By encoding its working instructions on its own DNA, the cell will be autonomous. Proliferation is carried out by a set of modules encoded on the DNA. These modules will serve to replicate the DNA itself, to replenish the gene expression machinery, and to stimulate growth and trigger division of the compartment. The goal of this thesis is to reconstitute compartment growth by cell-free gene expression of DNA encoding for phospholipid synthesis machinery.@en

Lipid membranes, nucleic acids, proteins, and metabolism are essential for modern cellular life. Synthetic systems emulating the fundamental properties of living cells must therefore be built upon these functional elements. In this work, phospholipid-producing enzymes encoded in a synthetic minigenome are cell-free expressed within liposome compartments. The de novo synthesized metabolic pathway converts precursors into a variety of lipids, including the constituents of the parental liposome. Balanced production of phosphatidylethanolamine and phosphatidylglycerol is realized, owing to transcriptional regulation of the activity of specific genes combined with a metabolic feedback mechanism. Fluorescence-based methods are developed to image the synthesis and membrane incorporation of phosphatidylserine at the single liposome level. Our results provide experimental evidence for DNA-programmed membrane synthesis in a minimal cell model. Strategies are discussed to alleviate current limitations toward effective liposome growth and self-reproduction.

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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.

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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.

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