Periodic Reporting for period 4 - PCELLS (Synthetic Cellularity via Protocell Design and Chemical Construction) Período documentado: 2022-01-01 hasta 2023-06-30 Resumen del contexto y de los objetivos generales del proyecto This research project aims to establish an unexplored frontier in protolife research that is based on novel conceptual and experimental advances in the design and construction of rudimentary forms of synthetic cell-like ensembles (protocells). Understanding how inanimate matter can be transformed into living matter is one of the major unsolved scientific problems of our time. Our overall goal is pioneer foundational steps towards minimal representations of synthetic cells as expressed through the fabrication of integrated, quasi-autonomous protocell phenotypes and communities. In this regard, the overall objectives are: (i) to advance a new area of synthetic protocell (bio)engineering that encompasses the design and construction of an inventory of novel protocell traits (protocell phenotypes), which exhibit increasing levels of complexity, functionality and primitive systems autonomy when compared with existing protocell models; and (ii) to explore higher-order behaviour (multi-compartmentalization, communication, signaling, predation, cooperation) within interacting protocell populations and communities (protocell ecosystems) as an unprecedented step towards synthetic protocell consortia and compartmentalized colloidal objects capable of novel collective and emergent properties. We expect our work to spearhead new advances in “Protolife Technologies” focused on the ex novo synthesis of minimal life constructs, and provide novel opportunities in bioinspired micro-storage and delivery, micro-reactor technologies, cytomimetic engineering, and the development of integrated constructs for diverse procedures in synthetic biology. Ultimately, our long-term goal is to achieve autonomous synthetic systems based on the functional self-integration of active matter. This would represent a groundbreaking step towards understanding and bridging the transition from non-living to living matter, and open up a new area of science with profound fundamental and applied consequences. Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha Work and outcomes to date against objectives. With regard to each theme, the following achievements have been made: Theme 1: Functional complexity in protocell phenotypes. With regard to Project 1.1 (Photosynthetic Protocells), we demonstrated that controlling the surface potential of coacervate micro-droplets can be used to capture intact plant chloroplasts to produce a membrane-free protocell model capable of light-induced electron transport. The chloroplasts retain their structural and functional integrity after immobilization within the coacervate phase and remain photoactive with a similar half-life of 3 days compared with isolated chloroplasts in buffer. [Kumar P B V V S et al; Chem. Commun., 54, 3594-3597 (2018)). With regard to Project 1.2 (Motile Protocells), we have described the design and construction of catalase-containing organoclay/DNA semipermeable microcapsules, which in the presence of hydrogen peroxide exhibit enzyme-powered oxygen gas bubble-dependent buoyancy. We determined the optimum conditions for single/multiple bubble generation per microcapsule, monitored the protocell velocities and resilience, and used remote magnetic guidance to establish reversible changes in buoyancy. Co-encapsulation of catalase and glucose oxidase was exploited to establish a spatiotemporal response to antagonistic bubble generation and depletion to produce protocells capable of sustained oscillatory vertical movement. Theme 2: Protocell self-structuring and metamorphosis. With regard to Project 2.1 (Self-structuration in Proteinosome-based Protocells), we have described the fabrication of stable colloidosomes derived from water-in-water Pickering-like emulsions produced by addition of fluorescent amine-modified polystyrene latex beads to an aqueous two-phase system. A key significance of our work is that colloidosomes produced specifically in all-water systems could offer new opportunities in microencapsulation and the bottom-up construction of synthetic protocells. [Douliez J-P, et al, Angew. Chemie. Int. Ed. 57, 7780-7784 (2018). With regard to Project 2.2 (Self-Transformation in Coacervate Micro-Droplets) we have used coacervate droplets to mimic the dynamical organization of membrane-free organelles in living systems. We reported the formation and photo-switchable behaviour of light-responsive coacervate droplets prepared from mixtures of double-stranded DNA and an azobenzene cation.. Sequestration and release of captured oligonucleotides followed the dynamics of phase separation such that light-activated transfer, mixing, hybridization and trafficking of the oligonucleotides was controlled in binary populations of the droplets. These results opened perspectives for the spatiotemporal control of DNA coacervates and provided a step towards the dynamic regulation of synthetic protocells. [Martin N, et al. Angew. Chem. Int. Ed. 58, 14594-14598 (2019)].Theme 3: Multi-compartmentalization: towards protocell endosymbiosis.. With regard to Projects 3.1 and 3.2 (Reaction Networks within Nested Proteinosomes; and Nucleated Protocells via Vesicle/Proteinosome Integration), we implemented the spontaneous capture and confinement of glucose oxidase (GOx)-containing proteinosomes in pH-sensitive horseradish peroxidase-containing fatty acid micelle coacervate micro-droplets as a facile route to multi-compartmentalized host-guest protocells capable of antagonistic modes of chemical and structural coupling. Co-encapsulation of antagonistic enzymes (urease and GOx) within the proteinosomes produced a sequence of self-induced capture and host-guest reconfigurationTheme 4: Collective behaviour in dispersed protocell populations. With regard to Project 4.1 (Chemical Signalling in Acoustically Ordered Protocell Arrays) we have developed the acoustic trapping method to pattern chloroplast-containing coacervate micro-droplets (see Theme 1; Project, Photosynthetic Protocells) into periodic 2D arrays with a controllable spacing, well-defined droplet size and narrow number distribution of the guest organelles. This has enabled an array of photosynthetic protocells to be routinely constructed. [Kumar P B V V S et al; Chem. Commun., 54, 3594-3597 (2018)). We have also used the acoustic trapping method to establish arrays of catalytic protocells (see section 2, below). [Gobbo P et al; Nature Commun. 11, 41 (2020)]With regard to Project 4.2 (Predator-Prey Behaviour in Protocell Communities), we exploited the dynamics of a model protocell community to modulate the function and higher-order behaviour of mixed populations of bioinorganic protocells in response to a process of artificial phagocytosis. Specifically, catalase, lipase or alkaline phosphatase-filled colloidosomes were used to trigger phagocytosis-induced buoyancy, membrane reconstruction or hydrogelation, respectively, within magnetic droplets. Taken together these results highlighted the potential for exploiting surface-contact interactions between different membrane-bounded droplets to transfer and co-locate discrete chemical packages (artificial “organelles”) in communities of synthetic protocells. [Rodríguez-Arco L et al., Angew. Chemie. Int. Ed. 58, 6333-6337 (2019).] Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha) We have developed the acoustic trapping method to pattern chloroplast-containing coacervate micro-droplets (see Theme 1; Project, Photosynthetic Protocells) into periodic 2D arrays with a controllable spacing, well-defined droplet size and narrow number distribution of the guest organelles. This has enabled an array of photosynthetic protocells to be routinely constructed. From a wider perspective, given the current need for clean energy and carbon dioxide remediation, chloroplasts are an attractive photoactive target to encapsulate or stabilize in ex situ environments. Indeed, chloroplasts have been stabilized in inert silica-based inert matrices for use as artificial photosynthetic bioreactors, and the extension to dispersed soft matter systems such as coacervates could provide new opportunities in flow-based systems and patterned droplet arrays. In the latter case, the ability to spontaneously produce uniform arrays of droplet-isolated chloroplasts might provide a novel route to photosynthetically active micro-chips for biomimetic water splitting. [Kumar P B V V S et al; Chem. Commun., 54, 3594-3597 (2018)).