Controlling Synthetic Cell-Cell Communication

Synthetic cells, which mimic cellular function within a minimal compartment, are finding wide application, for instance in studying cellular communication and as delivery devices to living cells. However, to fully realise the potential of synthetic cells, control of their function is vital. An array of tools has already been developed to control the communication of synthetic cells to neighbouring synthetic cells or living cells. These tools use either chemical inputs, such as small molecules, or physical inputs, such as light. Here, we examine these current methods of controlling synthetic cell communication and consider alternative mechanisms for future use.

Controlled exchange of protein and nucleic acid signals from and between synthetic minimal cells

Synthetic minimal cells are a class of small liposome bioreactors that have some, but not all functions of live cells. Here, we report a critical step towards the development of a bottom-up minimal cell: cellular export of functional protein and RNA products. We used cell penetrating peptide tags to translocate payloads across a synthetic cell vesicle membrane. We demonstrated efficient transport of active enzymes, and transport of nucleic acid payloads by RNA binding proteins. We investigated influence of a concentration gradient alongside other factors on the efficiency of the translocation, and we show a method to increase product accumulation in one location. We demonstrate the use of this technology to engineer molecular communication between different populations of synthetic cells, to exchange protein and nucleic acid signals. The synthetic minimal cell production and export of proteins or nucleic acids allows experimental designs that approach the complexity and relevancy of natural biological systems.

A synthetic membrane shaper for controlled liposome deformation

Shape defines the structure and function of cellular membranes. In cell division, the cell membrane deforms into a dumbbell shape, while organelles such as the autophagosome exhibit stomatocyte shapes. Bottom-up in vitro reconstitution of protein machineries that stabilize or resolve the membrane necks in such deformed liposome structures is of considerable interest to characterize their function. Here we develop a DNA-nanotechnology-based approach that we call Synthetic Membrane Shaper (SMS), where cholesterol-linked DNA structures attach to the liposome membrane to reproducibly generate high yields of stomatocytes and dumbbells. In silico simulations confirm the shape-stabilizing role of the SMS. We show that the SMS is fully compatible with protein reconstitution by assembling bacterial divisome proteins (DynaminA, FtsZ:ZipA) at the catenoidal neck of these membrane structures. The SMS approach provides a general tool for studying protein binding to complex membrane geometries that will greatly benefit synthetic cell research.

Quantification of microbial robustness

Stable cell performance in a fluctuating environment is essential for sustainable bioproduction and synthetic cell functionality; however, microbial robustness is rarely quantified. Here, we describe a high-throughput strategy for quantifying robustness of multiple cellular functions and strains in a perturbation space. We evaluated quantifications theory on experimental data and concluded that the mean-normalized Fano factor allowed accurate, reliable, and standardized quantification. Our methodology applied to perturbations related to lignocellulosic bioethanol production showed that Saccharomyces cerevisiae Ethanol Red exhibited both higher and more robust growth rates than CEN.PK and PE-2, while a more robust product yield traded off for lower mean levels. The methodology validated that robustness is function-specific and characterized by positive and negative function-specific trade-offs. Systematic quantification of robustness to end-use perturbations will be important to analyze and construct robust strains with more predictable functions.

Chemical communication at the synthetic cell/living cell interface

Although the complexity of synthetic cells has continued to increase in recent years, chemical communication between protocell models and living organisms remains a key challenge in bottom-up synthetic biology and bioengineering. In this Review, we discuss how communication channels and modes of signal processing can be established between living cells and cytomimetic agents such as giant unilamellar lipid vesicles, proteinosomes, polysaccharidosomes, polymer-based giant vesicles and membrane-less coacervate micro-droplets. We describe three potential modes of chemical communication in consortia of synthetic and living cells based on mechanisms of distributed communication and signal processing, physical embodiment and nested communication, and network-based contact-dependent communication. We survey the potential for applying synthetic cell/living cell communication systems in biomedicine, including the in situ production of therapeutics and development of new bioreactors. Finally, we present a short summary of our findings.

Synthetic Cell as a Platform for Understanding Membrane-Membrane Interactions

In the pursuit of understanding life, model membranes made of phospholipids were envisaged decades ago as a platform for the bottom-up study of biological processes. Micron-sized lipid vesicles have gained great acceptance as their bilayer membrane resembles the natural cell membrane. Important biological events involving membranes, such as membrane protein insertion, membrane fusion, and intercellular communication, will be highlighted in this review with recent research updates. We will first review different lipid bilayer platforms used for incorporation of integral membrane proteins and challenges associated with their functional reconstitution. We next discuss different methods for reconstitution of membrane fusion and compare their fusion efficiency. Lastly, we will highlight the importance and challenges of intercellular communication between synthetic cells and synthetic cells-to-natural cells. We will summarize the review by highlighting the challenges and opportunities associated with studying membrane–membrane interactions and possible future research directions.

Philosophy of Technoscience: From Cis-Continental to Trans-Continental

The previous chapters explored how four (interacting and overlapping) continental approaches (dialectics, dialectical materialism, psychoanalysis and phenomenology) offer hints and guidance for coming to terms with the revolutionary dynamics and disruptive impact of contemporary technoscience. Hegelian dialectics provides a conceptual scaffold for developing a comprehensive view of the terrestrial system and even for addressing the Cambrian explosion currently unfolding in laboratories around the globe, as a result of technoscientific developments such as synthetic biology and CRISP-Cas9. Dialectical materialism likewise offers a conceptual framework for addressing the rapidly aggravating disruption of the metabolism between nature and global civilisation, and the ongoing convergence of biosphere and technosphere, exemplified by the synthetic cell. Francophone psychoanalysis, closely aligned with dialectical thinking, adds to our understanding of the specificity of technoscience, both as a practice and as a discourse, where technoscientific research emerges as a questionable vocation driven by a desire to control, but at the same time ostensibly out of control. The dialectical methodology of psychoanalysis was exemplified with the help of case histories, moreover, involving Majorana particles, gene drives, malaria mosquitoes and nude mice. The latter represent technoscientific commodities, exemplifying the assembly-line production of human-made organisms (the commodification of life as such). Subsequently, we demonstrated how Heideggerian phenomenology entails important methodological hints for understanding technoscientific artefacts against the backdrop of technoscience as a mobilising force and as a global enterprise. And finally, we outlined how Teilhard’s views on the genesis of consciousness, self-consciousness and hyperconsciousness retrieve the historical (dialectical) dimension of phenomenology, thus allowing us to assess the present as a global unfolding of the noosphere.

Min waves without MinC can pattern FtsA-FtsZ filaments on model membranes

Although the essential proteins that drive bacterial cytokinesis have been identified and reconstituted in vitro, the precise mechanisms by which they dynamically interact to enable symmetrical division are largely unknown. In Escherichia coli, cell division begins with the formation of a proto-ring composed of FtsZ and its membrane-tethering proteins FtsA and ZipA. In the broadly proposed molecular scenario for ring positioning, Min waves composed of MinD and MinE distribute the FtsZ-polymerization inhibitor MinC away from mid-cell, where the Z-ring can form. Therefore, MinC is believed to be an essential element connecting the Min and FtsZ systems. Here, by using cell-free gene expression on planar lipid membranes, we demonstrate that MinDE drive the formation of dynamic, antiphase patterns of FtsZ-FtsA co-filaments even in the absence of MinC. This behavior is also observed when the proteins are compartmentalized inside microdroplets. These results suggest that Z-ring positioning may be achieved with a more minimal set of proteins than previously envisaged, providing a fresh perspective about the role of MinC. Moreover, we propose that MinDE oscillations may constitute the minimal localization mechanism of an FtsA-FtsZ constricting ring in a prospective synthetic cell.