Dynamic Synthetic Cells Based on Liquid–Liquid Phase Separation

Living cells have long been a source of inspiration for chemists. Their capacity of performing complex tasks relies on the spatiotemporal coordination of matter and energy fluxes. Recent years have witnessed growing interest in the bottom-up construction of cell-like models capable of reproducing aspects of such dynamic organisation. Liquid–liquid phase-separation (LLPS) processes in water are increasingly recognised as representing a viable compartmentalisation strategy through which to produce dynamic synthetic cells. Herein, we highlight examples of the dynamic properties of LLPS used to assemble synthetic cells, including their biocatalytic activity, reversible condensation and dissolution, growth and division, and recent directions towards the design of higher-order structures and behaviour.     

Specific Spatial Localization of Actin and DNA in a Water/Water Microdroplet: Self‐Emergence of a Cell‐Like Structure

The effect of binary hydrophilic polymers on a pair of representative bio‐macromolecules in a living cell has been examined. The results showed that these bio‐macromolecules exhibited specific localization in cell‐sized droplets that were spontaneously formed through water/water microphase segregation under crowding conditions with coexisting polymers. In these experiments, a simple binary polymer system with poly(ethylene glycol) (PEG) and dextran (DEX) was used. Under the conditions of microphase segregation, DNA was entrapped within cell‐sized droplets rich in DEX. Similarly, F‐actin, linearly polymerized actin, was entrapped specifically within microdroplets rich in DEX, whereas G‐actin, a monomeric actin, was distributed evenly inside and outside these droplets. This study has been extended to a system with both F‐actin and DNA, and it was found that DNA molecules were localized separately from aligned F‐actin proteins to create microdomains inside microdroplets, reflecting the self‐emergence of a cellular morphology similar to a stage of cell division.     

A novel arterial Wick for gas–liquid phase separation

Gas–liquid phase separation under microgravity conditions or in small-scale fluidic systems represents a challenge for two-phase liquid-continuous systems. In this study, capillary channels formed by 3-mm diameter stretched stainless-steel springs coated with a commercial superhydrophobic coating are used to remove air bubbles from water. A single channel is capable of absorbing a stream of 3.7-mm diameter bubbles impinging on a small area of the channel at a rate of over 50 bubbles/s. High-permeability walls lead to fast individual absorption events (4 ms for 2.5-mm bubbles) where bubble collapse time is limited by the inertia of the surrounding liquid. A horizontal three-channel array has been shown capable of absorbing impinging bubbles from a sparger at superficial gas velocities of 0.03 m/s. The ultimate capacity of the 3-mm diameter channel is predicted to be much higher than what could be measured with the existing apparatus. © 2018 American Institute of Chemical Engineers AIChE J, 65: 1340–1354, 2019     

Lessons from a Minimal Genome: What Are the Essential Organizing Principles of a Cell Built from Scratch?

One of the primary challenges facing synthetic biology is reconstituting a living system from its component parts. A particularly difficult landmark is reconstituting a self-organizing system that can undergo autonomous chromosome compaction, segregation, and cell division. Here, we discuss how the syn3.0 minimal genome can inform us of the core self-organizing principles of a living cell and how these self-organizing processes can be built from the bottom up. The review underscores the importance of fundamental biology in rebuilding life from its molecular constituents.     

Polysulfone‐Activated Carbon Hybrid Particles for the Removal of BPA

Abstract

Polysulfone‐activated carbon (PSF‐AC) hybrid particles are prepared using a liquid–liquid phase separation technique. The PSF‐AC hybrid particles are then used for the removal of bisphenol A (BPA) from its aqueous solutions. The PSF‐AC hybrid particles have similar structure as the polysulfone beads; both of them have a skin layer outside and a porous structure inside. The adsorption ability increased significantly when activated carbons were embedded into the particles, and the hybrid particles showed relatively more adsorption ability when the activated carbon content was 50%. The adsorbed BPA to the hybrid particles could be effectively removed by ethanol, which indicates that the hybrid particles can be reused. It is easy to prepare the particle column using these kinds of hybrid particles and the hybrid particle column could more effectively remove BPA. The results suggested that the PSF‐AC hybrid particles have a potential to be used for the removal of BPA in environmental application.     

2′‐Deoxyribonucleoside Phosphoramidate Triphosphate Analogues as Alternative Substrates for E. coli Polymerase III

Thermostable bacterial polymerases like Taq, Therminator and Vent exo^? are able to perform DNA synthesis by using modified DNA precursors, a property that is exploited in several therapeutic and biotechnological applications. Viral polymerases are also known to accept modified substrates, and this has proven crucial in the development of antiviral therapies. However, non‐thermostable polymerases of bacterial origin, or engineered variants, that have similar substrate tolerance and could be used for synthetic biology purposes remain to be identified. We have identified the α subunit of Escherichia coli polymerase III (Pol III α) as a bacterial polymerase that is able to recognise and process as substrates several pyrophosphate‐modified dATP analogues in place of its natural substrate dATP for template‐directed DNA synthesis. A number of dATP analogues featuring a modified pyrophosphate group were able to serve as substrates during enzymatic DNA synthesis by Pol III α. Features such as the presence of potentially chelating chemical groups and the size and spatial flexibility of the chemical structure seem to be of major importance for the modified leaving group to play its role during the enzymatic reaction. In addition, we could establish that if the pyrophosphate group is altered, deoxynucleotide incorporation proceeds with an efficiency varying with the nature of the nucleobase. Our results represent a great step towards the achievement of a system of artificial DNA synthesis hosted by E. coli and involving the use of altered nucleotide precursors for nucleic acid synthesis.     

A framework for morphological evolution vis-à-vis phase transitions in polymer solutions

When a polymer crystallizes from solution, it is well known that the resulting morphology depends on whether any liquid–liquid phase separation (LLPS) has preceded crystallization. In addition to the dense morphology that results when crystallization occurs directly from a homogeneous solution, at least three other distinctly different morphologies are produced if crystallization follows LLPS. Although much work has been reported in this regard, a framework that can relate the path that a process might follow across a phase diagram to the consequent morphology is lacking. We report here the fundamental elements of a simple thermodynamic framework that serves to identify the driving forces that produce these different morphologies. It is based on identification of the nucleating phase, if any, in LLPS and coupling it with the domain in which nucleation of crystallization occurs. The essential elements of the framework for morphological evolution are demonstrated by relating the sequence of phase transitions to the morphology which can result in the crystallized polymer when a polymer solution is cooled from a homogeneous state at a high temperature. Four distinctly different morphologies are shown to evolve, depending on whether crystallization occurs (a) directly from a homogeneous solution (dense); (b) following binodal liquid–liquid phase separation, LLPS, with nucleation of the polymer-rich phase (GMP—globular microporous); (c) following spinodal LLPS (FMP—fibrillar microporous); or (d) following binodal LLPS with nucleation of the solvent-rich phase (CTMP—cell-tunnel microporous). An important implication of the framework is that a predictable sequence of “dense → GMP → FMP → CTMP → dense” morphologies has to arise with increase in overall polymer concentration in such solutions. The framework also serves to identify conditions, such as passage through specific temperature/concentration regions in the phase diagram, that would increase the likelihood of forming mixed or coexisting morphologies. However, it is still necessary to develop appropriate kinetic models to predict sizes of the morphological components within each of the four morphologies. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 73: 1343–1355, 1999     

How We Make DNA Origami

DNA origami has attracted substantial attention since its invention ten years ago, due to the seemingly infinite possibilities that it affords for creating customized nanoscale objects. Although the basic concept of DNA origami is easy to understand, using custom DNA origami in practical applications requires detailed know‐how for designing and producing the particles with sufficient quality and for preparing them at appropriate concentrations with the necessary degree of purity in custom environments. Such know‐how is not readily available for newcomers to the field, thus slowing down the rate at which new applications outside the field of DNA nanotechnology may emerge. To foster faster progress, we share in this article the experience in making and preparing DNA origami that we have accumulated over recent years. We discuss design solutions for creating advanced structural motifs including corners and various types of hinges that expand the design space for the more rigid multilayer DNA origami and provide guidelines for preventing undesired aggregation and on how to induce specific oligomerization of multiple DNA origami building blocks. In addition, we provide detailed protocols and discuss the expected results for five key methods that allow efficient and damage‐free preparation of DNA origami. These methods are agarose‐gel purification, filtration through molecular cut‐off membranes, PEG precipitation, size‐exclusion chromatography, and ultracentrifugation‐based sedimentation. The guide for creating advanced design motifs and the detailed protocols with their experimental characterization that we describe here should lower the barrier for researchers to accomplish the full DNA origami production workflow.