Biomolecular condensates are a promising platform for synthetic cell formation and constitute a potential missing link between the chemical and cellular stage of the origins of life. However, it has proven challenging to integrate complex reaction networks into biomolecular condensates, such as a cell-free in vitro transcription-translation (IVTT) system. Integrating IVTT into biomolecular condensates successfully is one precondition for condensation-based synthetic cell formation. Moreover, it would provide a proof of concept that biomolecular condensates are in principle compatible with the central dogma, one of the hallmarks of cellular life. Here, we have systemically investigated the compatibility of eight different (bio)molecular condensates with IVTT incorporation. Of these eight candidates, we have found that a green fluorescent protein-labeled, intrinsically disordered cationic protein (GFP-K₇₂) and single-stranded DNA (ssDNA) can form biomolecular condensates that are compatible with up to μM fluorescent protein expression. This shows that biomolecular condensates can indeed integrate complex reaction networks, confirming their use as synthetic cell platforms and hinting at a possible role in the origin of life.

The role of evolutionary theory at the origin of life is an extensively debated topic. The origin and early development of life is usually separated into a prebiotic phase and a protocellular phase, ultimately leading to the Last Universal Common Ancestor. Most likely, the Last Universal Common Ancestor was subject to Darwinian evolution, but the question remains to what extent Darwinian evolution applies to the prebiotic and protocellular phases. In this review, we reflect on the current status of evolutionary theory in origins of life research by bringing together philosophy of science, evolutionary biology, and empirical research in the origins field. We explore the various ways in which evolutionary theory has been extended beyond biology; we look at how these extensions apply to the prebiotic development of (proto)metabolism; and we investigate how the terminology from evolutionary theory is currently being employed in state-of-the-art origins of life research. In doing so, we identify some of the current obstacles to an evolutionary account of the origins of life, as well as open up new avenues of research.

Gene expression is a fundamental aspect in the construction of a minimal synthetic cell, and the use of chromosomes will be crucial for the integration and regulation of complex modules. Expression from chromosomes in vitro transcription and translation (IVTT) systems presents limitations, as their large size and low concentration make them far less suitable for standard IVTT reactions. Here, we addressed these challenges by optimizing lysate-based IVTT systems at low template concentrations. We then applied an active learning tool to adapt IVTT to chromosomes as template DNA. Further insights into the dynamic data set led us to adjust the previous protocol for chromosome isolation and revealed unforeseen trends pointing at limiting transcription kinetics in our system. The resulting IVTT conditions allowed a high template DNA efficiency for the chromosomes. In conclusion, our system shows a protein-to-chromosome ratio that moves closer to in vivo biology and represents an advancement toward chromosome-based synthetic cells.

Discussions on the status of definitions of life have long been dominated by a position known as definitional pessimism. Per the definitional pessimist, there is no point in trying to define life. This claim is defended in different ways, but one of the shared assumptions of all definitional pessimists is that our attempts to define life are attempts to provide a list of all necessary and sufficient conditions for something to count as alive. In other words, a definition of life is a strict, descriptive definition. Against this, several pragmatic alternatives have been put forward. On these pragmatic accounts, definitions of life are not strictly, but rather loosely descriptive. Their purpose is not to be true, but to be useful to scientists by guiding scientific practice. More recently, this position has come under attack for not being able to explain how our attempts to define life are connected to scientific progress within the biological sciences. Here, I argue to the contrary by showing how pragmatic definitions of life can be, and in fact are, conducive to scientific progress. Additionally, I show how the pragmatic account of definitions of life can be brought to bear upon our normative discussions involving definitions of life.

Giant unilamellar vesicles (GUVs) provide a powerful model compartment for synthetic cells. However, a key challenge is the incorporation of membrane proteins that allow for transport, energy transduction, compartment growth and division. Here, we have successfully incorporated the membrane protein complex SecYEG—the key bacterial translocase that is essential for the incorporation of newly synthesized membrane proteins—in GUVs. Our method consists of fusion of small unilamellar vesicles containing reconstituted SecYEG into GUVs, thereby forming SecGUVs. These are suitable for large-scale experiments while maintaining a high protein:lipid ratio. We demonstrate that incorporation of SecYEG into GUVs does not inhibit its translocation efficiency. Robust membrane protein functionalized proteo-GUVs are promising and flexible compartments for use in the formation and growth of synthetic cells.