Construction Of Artificial Cells Research Articles

Evaluation of polymersome permeability as a fundamental aspect towards the development of artificial cells and nanofactories

Polymersomes are synthetic vesicles with potential use in healthcare, chemical transformations in confined environment (nanofactories), and in the construction of artificial cells and organelles. In this framework, one of the most important features of such supramolecular structures is the permeability behavior allowing for selective control of mass exchange between the inner and outer compartments. The use of biological and synthetic nanopores in this regard is the most common strategy to impart permeability nevertheless, this typically requires fairly complex strategies to enable porosity. Yet, investigations concerning the permeability of polymer vesicles to different analytes still requires further exploration and, taking these considerations into account, we have detailed investigated the permeability behavior of a variety of polymersomes with regard to different analytes (water, protons, and rhodamine B) which were selected as models for solvents, ions, and small molecules. Polymersomes based on hydrophilic blocks of poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) or PEO (poly(ethylene oxide)) linked to the non-responsive blocks poly[N-(4-isopropylphenylacetamide)ethyl methacrylate] (PPPhA) or poly(methyl methacrylate) (PMMA), or to the stimuli pH-responsive block poly[2-(diisopropylamino)ethyl methacrylate] (PDPA) have been investigated. Interestingly, the produced PEO-based vesicles are notably larger than the ones produced using PHPMA-containing block copolymers. The experimental results reveal that all the vesicles are inherently permeable to some extent with permeability behavior following exponential profiles. Nevertheless, polymersomes based on PMMA as the hydrophobic component were demonstrated to be the least permeable to the small molecule rhodamine B as well as to water. The synthetic vesicles based on the pH-responsive PDPA block exhibited restrictive and notably slow proton permeability as attributed to partial chain protonation upon acidification of the medium. The dye permeability was evidenced to be much slower than ion or solvent diffusion, and in the case of pH-responsive assemblies, it was demonstrated to also depend on the ionic strength of the environment. These findings are understood to be highly relevant towards polymer selection for the production of synthetic vesicles with selective and time-dependent permeability, and it may thus contribute in advancing biomimicry and nanomedicine.

Protocells and Prebiotic Systems

The de novo synthesis and self-assembly of molecules to establish the framework of living systems is a key target in the field of systems chemistry. The construction of synthetic cellular systems from scratch is one important such route to achieve this goal. This Special Collection on Protocells and Prebiotic Systems, guest edited by Dora Tang and Avinash J. Patil, showcases some of the most exciting work done in this field today. (D.Tang photograph copyright MPI-CBG). The de novo synthesis of molecules and their self-assembly to establish the framework of living systems is a key goal of systems chemistry. One route to achieving this is by building synthetic cellular systems from scratch which provides a gateway to understand and control emergence that arises from an integration of functional molecules and biological nanostructures at various length scales. Progress in this area can have significant impact for unravelling the origins of life, synthetic biology and future biomedical applications whilst providing fundamental insights into the mechanisms of biological systems. This Special Collection on Protocells and Prebiotic Systems brings together new and exciting work that is taking place in this field and demonstrates the extraordinary breadth of contributions required to make progress in this area: From prebiotic chemistry, to a survey of the molecular parts which can be used to design and build programmable cell-like compartments, engineer catalytic microreactors or create higher ordered architectures, to the emergence of adaptable systems where chemical environments can tune the physicochemical properties of soft materials. Furthermore, the capability to assimilate these properties to mimic fundamental biological processes such as chemical signaling; motility; energy transduction or cell growth and division are ever increasing. Whilst these examples are crucial for establishing minimal living systems, there are exciting prospects to exploit synthetic-cell-like compartments in wide-ranging applications including biomimetic materials; novel stimuli-responsive delivery platforms; biocatalysis; environmental remediation; tissue engineering; regenerative medicine; living materials and in adaptive integration to biological cells. The opinions expressed in this publication are the view of the author(s) and do not necessarily reflect the opinions or views of ChemSystemsChem, the Publisher, Chemistry Europe, or the affiliated editors. Dora Tang is currently a research group leader at the MPI-CBG, Dresden (Germany). Her multidisciplinary lab reimagines and translates the physical phenemona which drive out of equilibrium processes in cells to novel, robust and dynamic synthetic cellular systems. The minimal systems address questions in origin of life and modern biology. (Photograph copyright MPI-CBG). Avinash J. Patil is a faculty and group leader at the Centre for Organized Matter Chemistry, School of Chemistry, University of Bristol (UK). His research interests cover the design and construction of artificial cells, smart microreactors, and functional hybrid nanomaterials by combining various aspects of biology, materials chemistry, and micro/nanofabrication techniques.

Ethane groups modified DNA nanopores to prolong the dwell time on live cell membranes for transmembrane transport.

Transmembrane transport, mostly relying on biological channels, is crucial for the metabolic processes of live cells including sensing, signaling, cellular communicating and molecular transport. Artificial biomimetic channels offer excellent opportunities for studying the mechanisms of the metabolic processes of live cells and promote the applications of gene transfection, drug delivery, and regulations of cellular communications. DNA nanopores can be designed flexibly and operated easily while maintaining good biocompatibility, offering a good candidate for applications in basic research. However, because of the small size and good biocompatibility of DNA nanopores, it is still difficult to form stable channels on the plasma membrane of live cells by DNA nanopores. As a result, it significantly limits the applications of DNA nanopores in vivo. Thus, in this work, we have constructed ethane-phosphorothioate (PPT) groups modified DNA nanopores (E-DNA nanopores) to simulate biological channels for the transmembrane transport of small molecules. The E-DNA nanopores were found to be more hydrophobic and stable to anchor at the plasma membrane of live cells for a longer time window for subsequent transmembrane transport after the modification of ethane-PPT groups. The membrane-spanning E-DNA nanopores with a longer dwell time window could inspire the design of new DNA nanostructures and expand their biological applications including biosensing and sequencing, construction of artificial cells and regulation of transmembrane transport.

Construction of Artificial Cell as an Autonomous Supramolecular Machine

Construction of Artificial Cell as an Autonomous Supramolecular Machine

Chemical-triggered artificial cell based on metal-organic framework

An artificial cell based on an inducible cell-free expression system and metal–organic frameworks (MOFs), which can be triggered by chemical signals, was proposed in this study. The DNA expression templates were spatiotemporally encapsulated in MOF nanoparticles by co-precipitation and then wrapped in bio-mimicking liposomes. The signal of acidic pH or glucose induced the decomposition of MOFs, exposing the DNAs to start the cell-free expression to produce desired biopharmaceutical proteins. Therefore, the transcription and translation process could be controlled by simple chemical signals. This study investigated an unexplored topic of using MOFs in the construction of artificial cells, which could open new possibilities for future synthetic biology studies and therapeutic applications.

Vesicle-based artificial cells: materials, construction methods and applications.

The construction of artificial cells with specific cell-mimicking functions helps to explore complex biological processes and cell functions in natural cell systems and provides an insight into the origins of life. Bottom-up methods are widely used for engineering artificial cells based on vesicles by the in vitro assembly of biomimetic materials. In this review, the design of artificial cells with a specific function is discussed, by considering the selection of synthetic materials and construction technologies. First, a range of biomimetic materials for artificial cells is reviewed, including lipid, polymeric and hybrid lipid/copolymer materials. Biomaterials extracted from natural cells are also covered in this part. Then, the formation of microscale, giant unilamellar vesicles (GUVs) is reviewed based on different technologies, including gentle hydration, electro-formation, phase transfer and microfluidic methods. Subsequently, applications of artificial cells based on single vesicles or vesicle networks are addressed for mimicking cell behaviors and signaling processes. Microreactors for synthetic biology and cell–cell communication are highlighted here as well. Finally, current challenges and future trends for the development and applications of artificial cells are described.

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.

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.

Optimization of the Inverted Emulsion Method for High-Yield Production of Biomimetic Giant Unilamellar Vesicles.

In the field of bottom‐up synthetic biology, lipid vesicles provide an important role in the construction of artificial cells. Giant unilamellar vesicles (GUVs), due to their membrane's similarity to natural biomembranes, have been widely used as cellular mimics. So far, several methods exist for the production of GUVs with the possibility to encapsulate biological macromolecules. The inverted emulsion‐based method is one such technique, which has great potential for rapid production of GUVs with high encapsulation efficiencies for large biomolecules. However, the lack of understanding of various parameters that affect production yields has resulted in sparse adaptation within the membrane and bottom‐up synthetic biology research communities. Here, we optimize various parameters of the inverted emulsion‐based method to maximize the production of GUVs. We demonstrate that the density difference between the emulsion droplets, oil phase, and the outer aqueous phase plays a crucial role in vesicle formation. We also investigated the impact that centrifugation speed/time, lipid concentration, pH, temperature, and emulsion droplet volume has on vesicle yield and size. Compared to conventional electroformation, our preparation method was not found to significantly alter the membrane mechanical properties. Finally, we optimize the parameters to minimize the time from workbench to microscope and in this way open up the possibility of time‐sensitive experiments. In conclusion, our findings will promote the usage of the inverted emulsion method for basic membrane biophysics studies as well as the development of GUVs for use as future artificial cells.

Microfluidic technologies for the bottom-up construction of artificial cells

Microfluidic technologies for the bottom-up construction of artificial cells

Engineering Synthetic Ribosomes In Vitro

Purely in vitro ribosome synthesis could provide a critical step towards unraveling the systems biology of ribosome biogenesis, constructing minimal cells from defined components, and engineering ribosomes with new functions. To that end, we have developed an integrated synthesis, assembly, and translation technology (termed iSAT) for the in vitro construction of Escherichia coli ribosomes in crude ribosome-free S150 extracts. iSAT allows for the simultaneous in vitro synthesis of ribosomal RNA (rRNA), assembly of rRNA with purified ribosomal proteins, and transcription and translation of a reporter protein as a measure of ribosome activity. Here, we describe the development of, and recent improvements to, the iSAT system. Key breakthroughs include refining extract preparation, tuning the transcriptional balance of rRNA and mRNA production, and adding crowding and reducing agents. In addition to technological advances, we show that iSAT makes possible the in vitro construction of modified ribosomes by introducing a 23S rRNA mutation that mediates resistance against clindamycin. We also show that iSAT can be used for studying ribosome assembly. We anticipate that iSAT will aid studies of ribosome biogenesis and the construction of artificial cells.

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.

2P155人工細胞の構築(1) : 巨大リポソーム内でのタンパク質の結晶化

2P155人工細胞の構築(1) : 巨大リポソーム内でのタンパク質の結晶化