Nucleated synthetic cells with genetically driven intercompartment communication.

Eukaryotic cells are characterized by multiple chemically distinct compartments, one of the most notable being the nucleus. Within these compartments, there is a continuous exchange of information, chemicals, and signaling molecules, essential for coordinating and regulating cellular activities. One of the main goals of bottom-up synthetic biology is to enhance the complexity of synthetic cells by establishing functional compartmentalization. There is a need to mimic autonomous signaling between compartments, which in living cells, is often regulated at the genetic level within the nucleus. This advancement is key to unlocking the potential of synthetic cells as cell models and as microdevices in biotechnology. However, a technological bottleneck exists preventing the creation of synthetic cells with a defined nucleus-like compartment capable of genetically programmed intercompartment signaling events. Here, we present an approach for creating synthetic cells with distinct nucleus-like compartments that can encapsulate different biochemical mixtures in discrete compartments. Our system enables in situ protein expression of membrane proteins, enabling autonomous chemical communication between nuclear and cytoplasmic compartments, leading to downstream activation of enzymatic pathways within the cell.

Biomimetic behaviors in hydrogel artificial cells through embedded organelles.

Artificial cells are biomimetic structures formed from molecular building blocks that replicate biological processes, behaviors, and architectures. Of these building blocks, hydrogels have emerged as ideal, yet underutilized candidates to provide a gel-like chassis in which to incorporate both biological and nonbiological componentry which enables the replication of cellular functionality. Here, we demonstrate a microfluidic strategy to assemble biocompatible cell-sized hydrogel-based artificial cells with a variety of different embedded functional subcompartments, which act as engineered synthetic organelles. The organelles enable the recreation of increasingly biomimetic behaviors, including stimulus-induced motility, content release through activation of membrane-associated proteins, and enzymatic communication with surrounding bioinspired compartments. In this way, we showcase a foundational strategy for the bottom-up construction of hydrogel-based artificial cell microsystems which replicate fundamental cellular behaviors, paving the way for the construction of next-generation biotechnological devices.

Stimuli-responsive vesicles as distributed artificial organelles for bacterial activation.

Intercellular communication is a hallmark of living systems. As such, engineering artificial cells that possess this behavior has been at the heart of activities in bottom-up synthetic biology. Communication between artificial and living cells has potential to confer novel capabilities to living organisms that could be exploited in biomedicine and biotechnology. However, most current approaches rely on the exchange of chemical signals that cannot be externally controlled. Here, we report two types of remote-controlled vesicle-based artificial organelles that translate physical inputs into chemical messages that lead to bacterial activation. Upon light or temperature stimulation, artificial cell membranes are activated, releasing signaling molecules that induce protein expression in Escherichia coli. This distributed approach differs from established methods for engineering stimuli-responsive bacteria. Here, artificial cells (as opposed to bacterial cells themselves) are the design unit. Having stimuli-responsive elements compartmentalized in artificial cells has potential applications in therapeutics, tissue engineering, and bioremediation. It will underpin the design of hybrid living/nonliving systems where temporal control over population interactions can be exerted.

Implanted synthetic cells trigger tissue angiogenesis through de novo production of recombinant growth factors.

Progress in bottom-up synthetic biology has stimulated the development of synthetic cells (SCs), autonomous protein-manufacturing particles, as dynamic biomimetics for replacing diseased natural cells and addressing medical needs. Here, we report that SCs genetically encoded to produce proangiogenic factors triggered the physiological process of neovascularization in mice. The SCs were constructed of giant lipid vesicles and were optimized to facilitate enhanced protein production. When introduced with the appropriate genetic code, the SCs synthesized a recombinant human basic fibroblast growth factor (bFGF), reaching expression levels of up to 9⋅106 protein copies per SC. In culture, the SCs induced endothelial cell proliferation, migration, tube formation, and angiogenesis-related intracellular signaling, confirming their proangiogenic activity. Integrating the SCs with bioengineered constructs bearing endothelial cells promoted the remodeling of mature vascular networks, supported by a collagen-IV basement membrane-like matrix. In vivo, prolonged local administration of the SCs in mice triggered the infiltration of blood vessels into implanted Matrigel plugs without recorded systemic immunogenicity. These findings emphasize the potential of SCs as therapeutic platforms for activating physiological processes by autonomously producing biological drugs inside the body.

Protocells Capable of Generating a Cytoskeleton-like Structure from intracellular Membrane-active Artificial Organelles.

The intricate nature of eukaryotic cells with differently viscous intracellular compartments provides (membrane-active) enzymes to trigger time- and concentration-dependent processes in the intra-/extracellular matrix. Herein, we capitalize on membrane-active artificial organelles (AOs) to develop fluidic and stable proteinaceous membrane-based protocells. AOs in protocells induce the self-assembly of oligopeptides into an artificial cytoskeleton that underline their influence on the structure and functionality of protocells. A series of microscopical tools is used to validate the intracellular assembly and distribution of cytoskeleton, the changing protocells morphology, and AOs inclusion within cytoskeletal growth. Thus, the dynamics, diffusion and viscosity of intracellular components in the presence of cytoskeleton are evaluated by fluorescence tools and enzymatic assay. Membrane-active alkaline phosphatase in polymersomes as AOs fulfills the requirements of biomimetic eukaryotic cells to trigger intracellular environment, mobility, viscosity, diffusion and enzymatic activity itself as well as high mechanical stability and high membrane fluidity of protocells. Thus membrane-active AOs in protocells thoroughly provide a variable reaction space in a changing intracellular environment and underline their regulatory role in the fabrication of complex protocell architectures and functions. This study demonstrates an important contribution to effective biomimicry of cell-like structures, shapes and functions.

Bridging the Gap between Nonliving Matter and Cellular Life.

A cell, the fundamental unit of life, contains the requisite blueprint information necessary to survive and to build tissues, organs, and systems, eventually forming a fully functional living creature. A slight structural alteration can result in data misprinting, throwing the entire life process off balance. Advances in synthetic biology and cell engineering enable the predictable redesign of biological systems to perform novel functions. Individual functions and fundamental processes at the core of the biology of cells can be investigated by employing a synthetically constrained micro or nanoreactor. However, constructing a life-like structure from nonliving building blocks remains a considerable challenge. Chemical compartments, cascade signaling, energy generation, growth, replication, and adaptation within micro or nanoreactors must be comparable with their biological counterparts. Although these reactors currently lack the power and behavioral sophistication of their biological equivalents, their interface with biological systems enables the development of hybrid solutions for real-world applications, such as therapeutic agents, biosensors, innovative materials, and biochemical microreactors. This review discusses the latest advances in cell membrane-engineered micro or nanoreactors, as well as the limitations associated with high-throughput preparation methods and biological applications for the real-time modulation of complex pathological states.

Light-Harvesting Artificial Cells Containing Cyanobacteria for CO2 Fixation and Further Metabolism Mimicking.

The bottom-up constructed artificial cells help to understand the cell working mechanism and provide the evolution clues for organisms. The energy supply and metabolism mimicry are the key issues in the field of artificial cells. Herein, an artificial cell containing cyanobacteria capable of light harvesting and carbon dioxide fixation is demonstrated to produce glucose molecules by converting light energy into chemical energy. Two downstream "metabolic" pathways starting from glucose molecules are investigated. One involves enzyme cascade reaction to produce H2 O2 (assisted by glucose oxidase) first, followed by converting Amplex red to resorufin (assisted by horseradish peroxidase). The other pathway is more biologically relevant. Glucose molecules are dehydrogenated to transfer hydrogens to nicotinamide adenine dinucleotide (NAD+ ) for the production of nicotinamide adenine dinucleotide hydride (NADH) molecules in the presence of glucose dehydrogenase. Further, NADH molecules are oxidized into NAD+ by pyruvate catalyzed by lactate dehydrogenase, meanwhile, lactate is obtained. Therefore, the cascade cycling of NADH/NAD+ is built. The artificial cells built here pave the way for investigating more complicated energy-supplied metabolism inside artificial cells.

Cell-Like Capsules with "Smart" Compartments.

Eukaryotic cells have inner compartments (organelles), each with distinct properties and functions. One mimic of this architecture, based on biopolymers, is the multicompartment capsule (MCC). Here, MCCs in which the inner compartments are chemically unique and "smart," i.e., responsive to distinct stimuli in an orthogonal manner are created. Specifically, one compartment alone is induced to degrade when the MCC is contacted with an enzyme while other compartments remain unaffected. Similarly, just one compartment gets degraded upon contact with reactive oxygen species generated from hydrogen peroxide (H2 O2 ). And thirdly, one compartment alone is degraded by an external, physical stimulus, namely, by irradiating the MCC with ultraviolet (UV) light. All these specific responses are achieved without resorting to complicated chemistry to create the compartments: the multivalent cation used to crosslink the biopolymer alginate (Alg) is simply altered. Compartments of Alg crosslinked by Ca2+ are shown to be sensitive to enzymes (alginate lyases) but not to H2 O2 or UV, whereas the reverse is the case with Alg/Fe3+ compartments. These results imply the ability to selectively burst open a compartment in an MCC "on-demand" (i.e., as and when needed) and using biologically relevant stimuli. The results are then extended to a sequential degradation, where compartments in an MCC are degraded one after another, leaving behind an empty MCC lumen. Collectively, this work advances the MCC as a platform that not only emulates key features of cellular architecture, but can also begin to capture rudimentary cell-like behaviors.

Available strategies for improving the biosynthesis of surfactin: a review.

Surfactin is an excellent biosurfactant with a wide range of application prospects in many industrial fields. However, its low productivity and high cost have largely limited its commercial applications. In this review, the pathways for surfactin synthesis in Bacillus strains are summarized and discussed. Further, the latest strategies for improving surfactin production, including: medium optimization, genome engineering methods (rational genetic engineering, genome reduction, and genome shuffling), heterologous synthesis, and the use of synthetic biology combined with metabolic engineering approaches to construct high-quality artificial cells for surfactin production using xylose, are described. Finally, the prospects for improving surfactin synthesis are discussed in detail.

Chemical Systems for Wetware Artificial Life: Selected Perspectives in Synthetic Cell Research.

The recent and important advances in bottom-up synthetic biology (SB), in particular in the field of the so-called "synthetic cells" (SCs) (or "artificial cells", or "protocells"), lead us to consider the role of wetware technologies in the "Sciences of Artificial", where they constitute the third pillar, alongside the more well-known pillars hardware (robotics) and software (Artificial Intelligence, AI). In this article, it will be highlighted how wetware approaches can help to model life and cognition from a unique perspective, complementary to robotics and AI. It is suggested that, through SB, it is possible to explore novel forms of bio-inspired technologies and systems, in particular chemical AI. Furthermore, attention is paid to the concept of semantic information and its quantification, following the strategy recently introduced by Kolchinsky and Wolpert. Semantic information, in turn, is linked to the processes of generation of "meaning", interpreted here through the lens of autonomy and cognition in artificial systems, emphasizing its role in chemical ones.

The Use of Cell-free Protein Synthesis to Push the Boundaries of Synthetic Biology.

Cell-free protein synthesis is emerging as a powerful tool to accelerate the progress of synthetic biology. Notably, cell-free systems that harness extracted synthetic machinery of cells can address many of the issues associated with the complexity and variability of living systems. In particular, cell-free systems can be programmed with various configurations of genetic information, providing great flexibility and accessibility to the field of synthetic biology. Empowered by recent progress, cell-free systems are now evolving into artificial biological systems that can be tailored for various applications, including on-demand biomanufacturing, diagnostics, and new materials design. Here, we review the key developments related to cell-free protein synthesis systems, and discuss the future directions of these promising technologies.

Building a synthetic mechanosensitive signaling pathway in compartmentalized artificial cells.

To date, reconstitution of one of the fundamental methods of cell communication, the signaling pathway, has been unaddressed in the bottom-up construction of artificial cells (ACs). Such developments are needed to increase the functionality and biomimicry of ACs, accelerating their translation and application in biotechnology. Here, we report the construction of a de novo synthetic signaling pathway in microscale nested vesicles. Vesicle-cell models respond to external calcium signals through activation of an intracellular interaction between phospholipase A2 and a mechanosensitive channel present in the internal membranes, triggering content mixing between compartments and controlling cell fluorescence. Emulsion-based approaches to AC construction are therefore shown to be ideal for the quick design and testing of new signaling networks and can readily include synthetic molecules difficult to introduce to biological cells. This work represents a foundation for the engineering of multicompartment-spanning designer pathways that can be utilized to control downstream events inside an AC, leading to the assembly of micromachines capable of sensing and responding to changes in their local environment.

A Modular, Dynamic, DNA-Based Platform for Regulating Cargo Distribution and Transport between Lipid Domains.

Cell membranes regulate the distribution of biological machinery between phase-separated lipid domains to facilitate key processes including signaling and transport, which are among the life-like functionalities that bottom-up synthetic biology aims to replicate in artificial-cellular systems. Here, we introduce a modular approach to program partitioning of amphiphilic DNA nanostructures in coexisting lipid domains. Exploiting the tendency of different hydrophobic "anchors" to enrich different phases, we modulate the lateral distribution of our devices by rationally combining hydrophobes and by changing nanostructure size and topology. We demonstrate the functionality of our strategy with a bioinspired DNA architecture, which dynamically undergoes ligand-induced reconfiguration to mediate cargo transport between domains via lateral redistribution. Our findings pave the way to next-generation biomimetic platforms for sensing, transduction, and communication in synthetic cellular systems.

Synthetic Cells: From Simple Bio-Inspired Modules to Sophisticated Integrated Systems.

Bottom-up synthetic biology is the science of building systems that mimic the structure and function of living cells from scratch. To do this, researchers combine tools from chemistry, materials science, and biochemistry to develop functional and structural building blocks to construct synthetic cell-like systems. The many strategies and materials that have been developed in recent decades have enabled scientists to engineer synthetic cells and organelles that mimic the essential functions and behaviors of natural cells. Examples include synthetic cells that can synthesize their own ATP using light, maintain metabolic reactions through enzymatic networks, perform gene replication, and even grow and divide. In this Review, we discuss recent developments in the design and construction of synthetic cells and organelles using the bottom-up approach. Our goal is to present representative synthetic cells of increasing complexity as well as strategies for solving distinct challenges in bottom-up synthetic biology.

Photoswitchable Molecular Communication between Programmable DNA-Based Artificial Membraneless Organelles.

Spatiotemporal organization of distinct biological processes in cytomimetic compartments is a crucial step towards engineering functional artificial cells. Mimicking controlled bi-directional molecular communication inside artificial cells remains a considerable challenge. Here we present photoswitchable molecular transport between programmable membraneless organelle-like DNA coacervates in a synthetic microcompartment. We use droplet microfluidics to fabricate membraneless non-fusing DNA coacervates by liquid-liquid phase separation in a water-in-oil droplet, and employ the interior DNA coacervates as artificial organelles to imitate intracellular communication via photo-regulated uni- and bi-directional transfer of biomolecules. Our results highlight a promising new route to assembly of multicompartment artificial cells with functional networks.

Mitochondria Encapsulation in Hydrogel-Based Artificial Cells as ATP Producing Subunits.

Artificial cells (ACs) aim to mimic selected structural and functional features of mammalian cells. In this context, energy generation is an important challenge to be addressed when self-sustained systems are desired. Here, mitochondria isolated from HepG2 cells are employed as natural subunits that facilitate chemically driven adenosine triphosphate (ATP) synthesis. The successful mitochondria isolation is confirmed by monitoring the preserved inner membrane potential, the respiration, and the ATP production ability. The encapsulation of the isolated mitochondria in gelatin-based hydrogels results in similar initial ATP production compared to mitochondria in solution with a sustained ATP production over 24 h. Furthermore, luciferase is coencapsulated with the mitochondria in gelatin-based particles to create ACs and employ the in situ produced ATP to drive the catalytic conversion of d-luciferin. The coencapsulation of luciferase-loaded liposomes with mitochondria in gelatin-based hydrogels is additionally explored where the encapsulation of mitochondria and liposomes resulted in clustering effects that are likely contributing to the functional performance of the active entities. Taken together, mitochondria show potential in cell mimicry to facilitate energy-dependent processes.

Manufacture of Multilayered Artificial Cell Membranes through Sequential Bilayer Deposition on Emulsion Templates.

Efforts to manufacture artificial cells that replicate the architectures, processes and behaviours of biological cells are rapidly increasing. Perhaps the most commonly reconstructed cellular structure is the membrane, through the use of unilamellar vesicles as models. However, many cellular membranes, including bacterial double membranes, nuclear envelopes, and organelle membranes, are multilamellar. Due to a lack of technologies available for their controlled construction, multilayered membranes are not part of the repertoire of cell-mimetic motifs used in bottom-up synthetic biology. To address this, we developed emulsion-based technologies that allow cell-sized multilayered vesicles to be produced layer-by-layer, with compositional control over each layer, thus enabling studies that would otherwise remain inaccessible. We discovered that bending rigidities scale with the number of layers and demonstrate inter-bilayer registration between coexisting liquid-liquid domains. These technologies will contribute to the exploitation of multilayered membrane structures, paving the way for incorporating protein complexes that span multiple bilayers.

Switching ON of Transcription-Translation System Using GUV Fusion by Co-supplementation of Calcium with Long-Chain Polyethylene Glycol.

Giant unilamellar vesicles (GUVs) have been used as a material for bottom-up synthetic biology. However, due to the semi-permeability of the membrane, the need for methods to fuse GUVs has increased. To this aim, methods that are simple and show low leakage during fusion are important. In this study, we report a method of GUV fusion by a divalent cation (Ca2+ ) enhanced with a long chain polyethylene glycol (PEG20k). The methods showed significant GUV fusion without leakage of internal components of GUVs and maintained cell-free transcription-translation ability inside the GUVs without external supplementation of macromolecules. We demonstrate that the Ca-PEG method can be applied for switching ON of transcription-translation in GUVs in a fusion-dependent manner. The method developed here can be applied to extend bottom-up synthetic biology and molecular robotics that use GUVs as a chassis.

Interfacing Living and Synthetic Cells as an Emerging Frontier in Synthetic Biology.

The construction of artificial cells from inanimate molecular building blocks is one of the grand challenges of our time. In addition to being used as simplified cell models to decipher the rules of life, artificial cells have the potential to be designed as micromachines deployed in a host of clinical and industrial applications. The attractions of engineering artificial cells from scratch, as opposed to re-engineering living biological cells, are varied. However, it is clear that artificial cells cannot currently match the power and behavioural sophistication of their biological counterparts. Given this, many in the synthetic biology community have started to ask: is it possible to interface biological and artificial cells together to create hybrid living/synthetic systems that leverage the advantages of both? This article will discuss the motivation behind this cellular bionics approach, in which the boundaries between living and non-living matter are blurred by bridging top-down and bottom-up synthetic biology. It details the state of play of this nascent field and introduces three generalised hybridisation modes that have emerged.

Non-Equilibrium Large-Scale Membrane Transformations Driven by MinDE Biochemical Reaction Cycles.

The MinDE proteins from E. coli have received great attention as a paradigmatic biological pattern-forming system. Recently, it has surfaced that these proteins do not only generate oscillating concentration gradients driven by ATP hydrolysis, but that they can reversibly deform giant vesicles. In order to explore the potential of Min proteins to actually perform mechanical work, we introduce a new model membrane system, flat vesicle stacks on top of a supported lipid bilayer. MinDE oscillations can repeatedly deform these flat vesicles into tubules and promote progressive membrane spreading through membrane adhesion. Dependent on membrane and buffer compositions, Min oscillations further induce robust bud formation. Altogether, we demonstrate that under specific conditions, MinDE self-organization can result in work performed on biomimetic systems and achieve a straightforward mechanochemical coupling between the MinDE biochemical reaction cycle and membrane transformation.