Yuval Elani

Vesicle-based artificial cells as chemical microreactors with spatially segregated reaction pathways

In the discipline of bottom-up synthetic biology, vesicles define the boundaries of artificial cells and are increasingly being used as biochemical microreactors operating in physiological environments. As the field matures, there is a need to compartmentalize processes in different spatial localities within vesicles, and for these processes to interact with one another. Here we address this by designing and constructing multi-compartment vesicles within which an engineered multi-step enzymatic pathway is carried out. The individual steps are isolated in distinct compartments, and their products traverse into adjacent compartments with the aid of transmembrane protein pores, initiating subsequent steps. Thus, an engineered signalling cascade is recreated in an artificial cellular system. Importantly, by allowing different steps of a chemical pathway to be separated in space, this platform bridges the gap between table-top chemistry and chemistry that is performed within vesicles.

Engineering multi-compartment vesicle networks

Vesicles serve important functions in the construction of artificial cells. They facilitate biochemical reactions by confining reactants and products in space, and delineate the boundaries of the protocell. They allow concentration gradients to form, and control the passage of molecules via embedded proteins. However, to date, manufacturing strategies have focussed on uni-compartmental structures, resulting in vesicles with homogenous internal content. This is in contrast to real cells which have spatial segregation of components and processes. We bridge this divide by fabricating networked multi-compartment vesicles. These were generated by encasing multiple water-in-oil droplets with an external bilayer, using a process of gravity-mediated phase-transfer. We were able to control the content of the compartments, and could define the vesicle architecture by varying the number of encased droplets. We demonstrated the bilayers were biologically functional by inserting protein channels, which facilitated material transfer between the internal compartments themselves, and between the compartments and their external environment. This paves the way for the construction of inter- and intra-vesicle communication networks. Importantly, multi-compartment vesicles allow the spatio-dynamic organisation seen in real cells to be introduced into artificial ones for the first time.

Engineering Compartmentalized Biomimetic Micro- and Nanocontainers

Compartmentalization of biological content and function is a key architectural feature in biology, where membrane bound micro- and nanocompartments are used for performing a host of highly specialized and tightly regulated biological functions. The benefit of compartmentalization as a design principle is behind its ubiquity in cells and has led to it being a central engineering theme in construction of artificial cell-like systems. In this review, we discuss the attractions of designing compartmentalized membrane-bound constructs and review a range of biomimetic membrane architectures that span length scales, focusing on lipid-based structures but also addressing polymer-based and hybrid approaches. These include nested vesicles, multicompartment vesicles, large-scale vesicle networks, as well as droplet interface bilayers, and double-emulsion multiphase systems (multisomes). We outline key examples of how such structures have been functionalized with biological and synthetic machinery, for example, to manufacture and deliver drugs and metabolic compounds, to replicate intracellular signaling cascades, and to demonstrate collective behaviors as minimal tissue constructs. Particular emphasis is placed on the applications of these architectures and the state-of-the-art microfluidic engineering required to fabricate, functionalize, and precisely assemble them. Finally, we outline the future directions of these technologies and highlight how they could be applied to engineer the next generation of cell models, therapeutic agents, and microreactors, together with the diverse applications in the emerging field of bottom-up synthetic biology.

Construction of membrane-bound artificial cells using microfluidics: a new frontier in bottom-up synthetic biology

The quest to construct artificial cells from the bottom-up using simple building blocks has received much attention over recent decades and is one of the grand challenges in synthetic biology. Cell mimics that are encapsulated by lipid membranes are a particularly powerful class of artificial cells due to their biocompatibility and the ability to reconstitute biological machinery within them. One of the key obstacles in the field centres on the following: how can membrane-based artificial cells be generated in a controlled way and in high-throughput? In particular, how can they be constructed to have precisely defined parameters including size, biomolecular composition and spatial organization? Microfluidic generation strategies have proved instrumental in addressing these questions. This article will outline some of the major principles underpinning membrane-based artificial cells and their construction using microfluidics, and will detail some recent landmarks that have been achieved.

Artificial cell mimics as simplified models for the study of cell biology

Living cells are hugely complex chemical systems composed of a milieu of distinct chemical species (including DNA, proteins, lipids, and metabolites) interconnected with one another through a vast web of interactions: this complexity renders the study of cell biology in a quantitative and systematic manner a difficult task. There has been an increasing drive towards the utilization of artificial cells as cell mimics to alleviate this, a development that has been aided by recent advances in artificial cell construction. Cell mimics are simplified cell-like structures, composed from the bottom-up with precisely defined and tunable compositions. They allow specific facets of cell biology to be studied in isolation, in a simplified environment where control of variables can be achieved without interference from a living and responsive cell. This mini-review outlines the core principles of this approach and surveys recent key investigations that use cell mimics to address a wide range of biological questions. It will also place the field in the context of emerging trends, discuss the associated limitations, and outline future directions of the field. Impact statement Recent years have seen an increasing drive to construct cell mimics and use them as simplified experimental models to replicate and understand biological phenomena in a well-defined and controlled system. By summarizing the advances in this burgeoning field, and using case studies as a basis for discussion on the limitations and future directions of this approach, it is hoped that this minireview will spur others in the experimental biology community to use artificial cells as simplified models with which to probe biological systems.

Constructing vesicle-based artificial cells with embedded living cells as organelle-like modules

There is increasing interest in constructing artificial cells by functionalising lipid vesicles with biological and synthetic machinery. Due to their reduced complexity and lack of evolved biochemical pathways, the capabilities of artificial cells are limited in comparison to their biological counterparts. We show that encapsulating living cells in vesicles provides a means for artificial cells to leverage cellular biochemistry, with the encapsulated cells serving organelle-like functions as living modules inside a larger synthetic cell assembly. Using microfluidic technologies to construct such hybrid cellular bionic systems, we demonstrate that the vesicle host and the encapsulated cell operate in concert. The external architecture of the vesicle shields the cell from toxic surroundings, while the cell acts as a bioreactor module that processes encapsulated feedstock which is further processed by a synthetic enzymatic metabolism co-encapsulated in the vesicle.

Light-triggered enzymatic reactions in nested vesicle reactors

Cell-sized vesicles have tremendous potential both as miniaturised pL reaction vessels and in bottom-up synthetic biology as chassis for artificial cells. In both these areas the introduction of light-responsive modules affords increased functionality, for example, to initiate enzymatic reactions in the vesicle interior with spatiotemporal control. Here we report a system composed of nested vesicles where the inner compartments act as phototransducers, responding to ultraviolet irradiation through diacetylene polymerisation-induced pore formation to initiate enzymatic reactions. The controlled release and hydrolysis of a fluorogenic β-galactosidase substrate in the external compartment is demonstrated, where the rate of reaction can be modulated by varying ultraviolet exposure time. Such cell-like nested microreactor structures could be utilised in fields from biocatalysis through to drug delivery.Matryoshka doll-like, nested vesicles, each containing a different ingredient to a chemical reaction, can serve as microreactors. Here, the authors developed a system in which mixing of the ingredients can be induced by irradiation with ultraviolet light.

Sculpting and fusing biomimetic vesicle networks using optical tweezers

Constructing higher-order vesicle assemblies has discipline-spanning potential from responsive soft-matter materials to artificial cell networks in synthetic biology. This potential is ultimately derived from the ability to compartmentalise and order chemical species in space. To unlock such applications, spatial organisation of vesicles in relation to one another must be controlled, and techniques to deliver cargo to compartments developed. Herein, we use optical tweezers to assemble, reconfigure and dismantle networks of cell-sized vesicles that, in different experimental scenarios, we engineer to exhibit several interesting properties. Vesicles are connected through double-bilayer junctions formed via electrostatically controlled adhesion. Chemically distinct vesicles are linked across length scales, from several nanometres to hundreds of micrometres, by axon-like tethers. In the former regime, patterning membranes with proteins and nanoparticles facilitates material exchange between compartments and enables laser-triggered vesicle merging. This allows us to mix and dilute content, and to initiate protein expression by delivering biomolecular reaction components.Assembly of higher-order artificial vesicles can unlock new applications. Here, the authors use optical tweezers to construct user-defined 2D and 3D architectures of chemically distinct vesicles and demonstrate inter-vesicle communication and light-enabled compartment merging.

Functionalizing cell-mimetic giant vesicles with encapsulated bacterial biosensors

The design of vesicle microsystems as artificial cells (bottom-up synthetic biology) has traditionally relied on the incorporation of molecular components to impart functionality. These cell mimics have reduced capabilities compared with their engineered biological counterparts (top-down synthetic biology), as they lack the powerful metabolic and regulatory pathways associated with living systems. There is increasing scope for using whole intact cellular components as functional modules within artificial cells, as a route to increase the capabilities of artificial cells. In this feasibility study, we design and embed genetically engineered microbes (Escherichia coli) in a vesicle-based cell mimic and use them as biosensing modules for real-time monitoring of lactate in the external environment. Using this conceptual framework, the functionality of other microbial devices can be conferred into vesicle microsystems in the future, bridging the gap between bottom-up and top-down synthetic biology.

Engineering thermoresponsive phase separated vesicles formed: Via emulsion phase transfer as a content-release platform

Elucidation of cholesterol insertion efficiency into phase-transfer vesicles enables the rational design of phase-separated membranes as thermally-responsive platforms for artificial cell construction.