Compartmentalizing Cell-Free Systems: Toward Creating Life-Like Artificial Cells and Beyond

Cho, Eunhee; Lu, Yuan

doi:10.1021/acssynbio.0c00433

Cited by 83 publications

(65 citation statements)

References 211 publications

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“…The construction of a compartmentalized system in synthetic eukaryotes is thus key in order to build more sophisticated synthetic cells for both fundamental biological investigations, and as promising novel biomaterial for healthcare applications. 13 − 15 …”

Section: Introductionmentioning

confidence: 99%

pH-Triggered Assembly of Endomembrane Multicompartments in Synthetic Cells

et al. 2021

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By using electrostatic interactions as driving force to assemble vesicles, the droplet-stabilized method was recently applied to reconstitute and encapsulate proteins, or compartments, inside giant unilamellar vesicles (GUVs) to act as minimal synthetic cells. However, the droplet-stabilized approach exhibits low production efficiency associated with the troublesome release of the GUVs from the stabilized droplets, corresponding to a major hurdle for the droplet-stabilized approach. Herein, we report the use of pH as a potential trigger to self-assemble droplet-stabilized GUVs (dsGUVs) by either bulk or droplet-based microfluidics. Moreover, pH enables the generation of compartmentalized GUVs with flexibility and robustness. By co-encapsulating pH-sensitive small unilamellar vesicles (SUVs), negatively charged SUVs, and/or proteins, we show that acidification of the droplets efficiently produces dsGUVs while sequestrating the co-encapsulated material. Most importantly, the pH-mediated assembly of dsGUVs significantly improves the production efficiency of free-standing GUVs (i.e., released from the stabilizing-droplets) compared to its previous implementation.

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Section: Introductionmentioning

confidence: 99%

pH-Triggered Assembly of Endomembrane Multicompartments in Synthetic Cells

et al. 2021

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“…Therefore, plasmids can be extracted from V. natriegens , C. glutamate and B. subtilis , respectively, and various expression vectors can be designed to further increase the expression of CFPS systems. In addition, studies on the metabolic pathways, gene circuits and diagnostic tools (Cho and Lu 2020 ; Lin et al 2021 ; Lin et al 2020a , b ; Yang and Lu 2020 ) in the E. coli CFPS system can be tried in the other three systems, and a diversified CFPS platform can be developed further to expand the application scope of the CFPS systems.…”

Section: Discussionmentioning

confidence: 99%

Development and comparison of cell-free protein synthesis systems derived from typical bacterial chassis

Zhang

Lin

Wang

et al. 2021

Bioresour. Bioprocess.

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Cell-free protein synthesis (CFPS) systems have become an ideal choice for pathway prototyping, protein production, and biosensing, due to their high controllability, tolerance, stability, and ability to produce proteins in a short time. At present, the widely used CFPS systems are mainly based on Escherichia coli strain. Bacillus subtilis, Corynebacterium glutamate, and Vibrio natriegens are potential chassis cells for many biotechnological applications with their respective characteristics. Therefore, to expand the platform of the CFPS systems and options for protein production, four prokaryotes, E. coli, B. subtilis, C. glutamate, and V. natriegens were selected as host organisms to construct the CFPS systems and be compared. Moreover, the process parameters of the CFPS system were optimized, including the codon usage, plasmid synthesis competent cell selection, plasmid concentration, ribosomal binding site (RBS), and CFPS system reagent components. By optimizing and comparing the main influencing factors of different CFPS systems, the systems can be optimized directly for the most influential factors to further improve the protein yield of the systems. In addition, to demonstrate the applicability of the CFPS systems, it was proved that the four CFPS systems all had the potential to produce therapeutic proteins, and they could produce the receptor-binding domain (RBD) protein of SARS-CoV-2 with functional activity. They not only could expand the potential options for in vitro protein production, but also could increase the application range of the system by expanding the cell-free protein synthesis platform.

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“…LLPS compartments have been proposed as primitive cell models and phase separation also produces membraneless organelles in modern organisms (Yoshizawa et al 2020). As such, there is significant interest in use of LLPS within synthetic biology, especially with the recent major development of cell-free enzymatic systems and processes applied to synthetic biology (Lu 2017;Tinafar et al 2019;Cho and Lu 2020). Here, we briefly review recent studies which seek to imbue biological processes into LLPS compartments and also discuss recent research utilizing phase separation in artificial cell construction.…”

Section: Llps Applied To Synthetic Biologymentioning

confidence: 99%

Connecting primitive phase separation to biotechnology, synthetic biology, and engineering

et al. 2021

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One aspect of the study of the origins of life focuses on how primitive chemistries assembled into the first cells on Earth and how these primitive cells evolved into modern cells. Membraneless droplets generated from liquid-liquid phase separation (LLPS) are one potential primitive cell-like compartment; current research in origins of life includes study of the structure, function, and evolution of such systems. However, the goal of primitive LLPS research is not simply curiosity or striving to understand one of life's biggest unanswered questions, but also the possibility to discover functions or structures useful for application in the modern day. Many applicational fields, including biotechnology, synthetic biology, and engineering, utilize similar phaseseparated structures to accomplish specific functions afforded by LLPS. Here, we briefly review LLPS applied to primitive compartment research and then present some examples of LLPS applied to biomolecule purification, drug delivery, artificial cell construction, waste and pollution management, and flavor encapsulation. Due to a significant focus on similar functions and structures, there appears to be much for origins of life researchers to learn from those working on LLPS in applicational fields, and vice versa, and we hope that such researchers can start meaningful cross-disciplinary collaborations in the future.

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Compartmentalizing Cell-Free Systems: Toward Creating Life-Like Artificial Cells and Beyond

Cited by 83 publications

References 211 publications

pH-Triggered Assembly of Endomembrane Multicompartments in Synthetic Cells

pH-Triggered Assembly of Endomembrane Multicompartments in Synthetic Cells

Development and comparison of cell-free protein synthesis systems derived from typical bacterial chassis

Connecting primitive phase separation to biotechnology, synthetic biology, and engineering

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