Periodic Reporting for period 1 - PROTOBAC (Engineering of complex protocells by micro-compartmentalization of living bacteria)
Período documentado: 2019-12-01 hasta 2021-11-30
Establishing true-to-life functionality in synthetic cells is a global grand challenge that traverses multiple fields including synthetic biology, bioengineering and origins of life research. However, advancing the spontaneous bottom-up construction of artificial cells with high organisational complexity and diverse functionality remains an unresolved issue at the interface between living and non-living matter.
The transitioning from inert capsule-based technologies to dynamical micro-compartmentalized entities capable of autonomous cytomimetic behaviour requires breakthrough advances in functional integration and on-board energization of multiplexed micro-systems. To date, the engineering of synthetic cellular systems (protocells) has been mainly approached using self-assembled vesicles, semi-permeable microcapsules and membrane-less or coated coacervate micro-droplets. These compartments provide a controllable medium for the encapsulation and exchange of biological and non-biological components that are experimentally selected to demonstrate single functions such as gene expression, enzyme catalysis and ribozyme activity within the synthetic cell models. Achieving high organisational and functional complexity in these constructs is methodological demanding due to difficulties in establishing sufficient compositional diversity and chemical complementarity by conventional methods of micro-compartmentalization under close-to-equilibrium conditions. These limitations restrict the structural and chemical complexity of current protocell models, inhibit the implementation of integrated componentry, and impede the development of energized cytomimetic systems.
To address this challenge, a living material assembly process based on the capture and on-site processing of spatially segregated bacterial colonies within individual coacervate micro-droplets is developed for the endogenous construction of membrane-bounded, molecularly crowded, compositionally, structurally and morphologically complex synthetic cells. The bacteriogenic protocells inherit diverse biological components, exhibit multi-functional cytomimetic properties and can be endogenously remodelled to include a spatially partitioned DNA/histone nucleus-like condensate, membranized water vacuoles and a self-supporting 3D network of F-actin proto-cytoskeletal filaments. The ensemble is biochemically energized by self-sustainable ATP production derived from implanted live E. coli cells to produce a cellular bionic system with amoeba-like external morphology and integrated life-like properties. Our results demonstrate a novel bacteriogenic strategy for the bottom-up construction of functional protoliving micro-devices and provide opportunities for the fabrication of new synthetic cell modules and augmented living/synthetic cell constructs with potential applications in engineered synthetic biology and biotechnology.
The transitioning from inert capsule-based technologies to dynamical micro-compartmentalized entities capable of autonomous cytomimetic behaviour requires breakthrough advances in functional integration and on-board energization of multiplexed micro-systems. To date, the engineering of synthetic cellular systems (protocells) has been mainly approached using self-assembled vesicles, semi-permeable microcapsules and membrane-less or coated coacervate micro-droplets. These compartments provide a controllable medium for the encapsulation and exchange of biological and non-biological components that are experimentally selected to demonstrate single functions such as gene expression, enzyme catalysis and ribozyme activity within the synthetic cell models. Achieving high organisational and functional complexity in these constructs is methodological demanding due to difficulties in establishing sufficient compositional diversity and chemical complementarity by conventional methods of micro-compartmentalization under close-to-equilibrium conditions. These limitations restrict the structural and chemical complexity of current protocell models, inhibit the implementation of integrated componentry, and impede the development of energized cytomimetic systems.
To address this challenge, a living material assembly process based on the capture and on-site processing of spatially segregated bacterial colonies within individual coacervate micro-droplets is developed for the endogenous construction of membrane-bounded, molecularly crowded, compositionally, structurally and morphologically complex synthetic cells. The bacteriogenic protocells inherit diverse biological components, exhibit multi-functional cytomimetic properties and can be endogenously remodelled to include a spatially partitioned DNA/histone nucleus-like condensate, membranized water vacuoles and a self-supporting 3D network of F-actin proto-cytoskeletal filaments. The ensemble is biochemically energized by self-sustainable ATP production derived from implanted live E. coli cells to produce a cellular bionic system with amoeba-like external morphology and integrated life-like properties. Our results demonstrate a novel bacteriogenic strategy for the bottom-up construction of functional protoliving micro-devices and provide opportunities for the fabrication of new synthetic cell modules and augmented living/synthetic cell constructs with potential applications in engineered synthetic biology and biotechnology.
Our projects is based on the co-capture and on-site processing of two spatially segregated bacterial colonies (Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa, PAO1 strain) that are co-associated with individual poly(diallyldimethylammonium chloride) (PDDA)/adenosine 5’-triphosphate (ATP) or uridine 5’-triphosphate (UTP) coacervate micro-droplets.
In situ lysis of the captured bacteria spontaneously gives rise to lipid membrane-coated protocells enclosing an extensive repertoire of functional biological components. We demonstrate that the bacteriogenic protocells are capable of complex processing such as the endogenous production of ATP via proto-metabolic activity (glycolysis) and inherit a sufficient complement of the bacterial gene expression machinery to implement in vitro transcription and translation.
To increase the level of internal structural organization, we use a combination of endogenous polynucleotide liquid-liquid phase separation, ATP-driven supramolecular protein assembly and hypotonicity to augment the synthetic cells with a spatially partitioned nucleus-like DNA/histone condensate, 3D network of F-actin proto-cytoskeletal filaments and osmotically responsive membrane-bounded water vacuoles, respectively.
As a step towards self-sustainable energization, we construct living/synthetic hybrids in which we exploit implanted live E. coli cells as surrogate mitochondria to increase and extend the endogenous production of ATP for enhancing kinase activity, glycolysis, in vitro gene expression and cytoskeletal assembly within the bacteriogenic protocells.
The final protoliving constructs adopt an amoeba-like external morphology and decreased membrane permeability due to on-site bacterial metabolism and growth to produce a cellular bionic system with integrated life-like properties.
Taken together, our results demonstrate a novel bacteriogenic strategy for the bottom-up construction of functional protocellular micro-devices and provide opportunities for the fabrication of new synthetic cell modules and augmented living/synthetic cell constructs with potential applications in engineered synthetic biology and biotechnology.
In situ lysis of the captured bacteria spontaneously gives rise to lipid membrane-coated protocells enclosing an extensive repertoire of functional biological components. We demonstrate that the bacteriogenic protocells are capable of complex processing such as the endogenous production of ATP via proto-metabolic activity (glycolysis) and inherit a sufficient complement of the bacterial gene expression machinery to implement in vitro transcription and translation.
To increase the level of internal structural organization, we use a combination of endogenous polynucleotide liquid-liquid phase separation, ATP-driven supramolecular protein assembly and hypotonicity to augment the synthetic cells with a spatially partitioned nucleus-like DNA/histone condensate, 3D network of F-actin proto-cytoskeletal filaments and osmotically responsive membrane-bounded water vacuoles, respectively.
As a step towards self-sustainable energization, we construct living/synthetic hybrids in which we exploit implanted live E. coli cells as surrogate mitochondria to increase and extend the endogenous production of ATP for enhancing kinase activity, glycolysis, in vitro gene expression and cytoskeletal assembly within the bacteriogenic protocells.
The final protoliving constructs adopt an amoeba-like external morphology and decreased membrane permeability due to on-site bacterial metabolism and growth to produce a cellular bionic system with integrated life-like properties.
Taken together, our results demonstrate a novel bacteriogenic strategy for the bottom-up construction of functional protocellular micro-devices and provide opportunities for the fabrication of new synthetic cell modules and augmented living/synthetic cell constructs with potential applications in engineered synthetic biology and biotechnology.
Our new living material assembly approach provides opportunities for the bottom-up construction of highly integrated synthetic cells and augmented living/synthetic cell constructs.
A critical aspect of the construction sequence is the spontaneous coacervate droplet-mediated capture and spatial segregation of E. coli and PA01 cells that together enable on-site processing and retention of diverse bacterial components for synthetic cell elaboration. Thus, using this strategy it should be possible not only to establish artificial organelles based on lysosomes, peroxisomes and storage granules within the bacteriogenic protocells, but also reconfigure the spatial organization of the bacterially derived components. For example, the spatial distribution of live E. coli and PA01 cells in the initial pre-former coacervate droplets can be modulated by changes in the coacervate composition.
By using engineered bacteria designed to deliver specialized components and biological processes for establishing robust metabolic networks and genetic circuitry in the bacteriogenic protocells, we expect the methodology to be responsive to high levels of programmability in future studies.
Finally, from a cellular bionic perspective, the potential for symbiosis in living/synthetic cell hybrids constructed from bacterially derived construction pathways could offer more complex modules for development in diagnostic and therapeutic areas of synthetic biology as well as in biomanufacturing and biotechnology in general.
Coupling these endogenous circuits to diagnostic functions towards cytokines, hormones and metabolites will be challenging, ultimately relying on the high capture efficiency of the internal coacervate matrix and appropriate design of the output signals via judicious engineering of the living/synthetic cell interface.
A critical aspect of the construction sequence is the spontaneous coacervate droplet-mediated capture and spatial segregation of E. coli and PA01 cells that together enable on-site processing and retention of diverse bacterial components for synthetic cell elaboration. Thus, using this strategy it should be possible not only to establish artificial organelles based on lysosomes, peroxisomes and storage granules within the bacteriogenic protocells, but also reconfigure the spatial organization of the bacterially derived components. For example, the spatial distribution of live E. coli and PA01 cells in the initial pre-former coacervate droplets can be modulated by changes in the coacervate composition.
By using engineered bacteria designed to deliver specialized components and biological processes for establishing robust metabolic networks and genetic circuitry in the bacteriogenic protocells, we expect the methodology to be responsive to high levels of programmability in future studies.
Finally, from a cellular bionic perspective, the potential for symbiosis in living/synthetic cell hybrids constructed from bacterially derived construction pathways could offer more complex modules for development in diagnostic and therapeutic areas of synthetic biology as well as in biomanufacturing and biotechnology in general.
Coupling these endogenous circuits to diagnostic functions towards cytokines, hormones and metabolites will be challenging, ultimately relying on the high capture efficiency of the internal coacervate matrix and appropriate design of the output signals via judicious engineering of the living/synthetic cell interface.