Regulation of spatiotemporal patterning in artificial cells by a defined protein expression system

Yoshida, Asami; Kohyama, Shunshi; Fujiwara, Keigi; Nishikawa, Saki; Doi, Nobuhide

doi:10.1039/c9sc02441g

Cited by 38 publications

(64 citation statements)

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“…Because of its easiness of handling and ability to express wide varieties of proteins, CFPS is widely applied to protein engineering including directed evolution of proteins and synthesis of cytotoxic proteins [ 2 , 3 , 4 , 5 , 6 , 7 ]. Since CFPS has the ability to reconstitute biochemical systems from DNA mixtures, it has been also featured as an important material for research toward rebuilding living cells in vitro [ 8 , 9 , 10 , 11 , 12 , 13 ]. As another application, CFPS has been utilized for protein folding studies [ 14 , 15 ], membrane insertion analysis [ 16 , 17 ], and elucidation of molecular machinery in living cells such as translational arrest [ 18 ].…”

Section: Introductionmentioning

confidence: 99%

A Relationship between NTP and Cell Extract Concentration for Cell-Free Protein Expression

Takahashi

Sato

Doi

et al. 2021

Life

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The cell-free protein synthesis (CFPS) that synthesizes mRNA and protein from a template DNA has been featured as an important tool to emulate living systems in vitro. However, an obstacle to emulate living cells by CFPS is the loss of activity in the case of usage of high concentration cell extracts. In this study, we found that a high concentration of NTP which inhibits in the case of lower concentration cell extract restored the loss of CFPS activity using high concentration cell extracts. The NTP restoration was independent of the energy regeneration system used, and NTP derivatives also restored the levels of CFPS using a high concentration cell extract. Experiments using dialysis mode of CFPS showed that continuous exchange of small molecule reduced levels of NTP requirement and improved reaction speed of CFPS using the high concentration of cell extract. These findings contribute to the development of a method to understand the condition of living cells by in vitro emulation, and are expected to lead to the achievement of the reconstitution of living cells from biomolecule mixtures.

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Section: Introductionmentioning

confidence: 99%

A Relationship between NTP and Cell Extract Concentration for Cell-Free Protein Expression

Takahashi

Sato

Doi

et al. 2021

Life

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“…In order to avoid quantity encapsulation fluctuations during simultaneous addition of various DNA into cell‐free systems, Doi and co‐workers simplified the tedious regulation of plasmid DNA by constructing DNA operon structure. [ 121 ] They also discovered that crowding environments modified Min protein binding onto the artificial cell membrane. [ 122 ]…”

Section: Dynamic Behaviorsmentioning

confidence: 99%

Emerging Advances of Cell‐Free Systems toward Artificial Cells

et al. 2020

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models have attracted considerable attentions in order to unravel the inherent complexity. Although engineering modern cells has achieved some progress, [2] the arbitrary elimination of genomic information could probably remove some distinguished characteristics (top-down strategy). Building chemical systems that resemble the structure and behaviors of living cells by stepwise integration of pivotal components is also conceivable (bottom-up strategy). [3] These synthetic systems with biomimetic cellularity provide conceptual pathway from inanimate matter to artificial life, which are often known as artificial cells. [4] Due to the lack of full comprehension about the molecular mechanism of living cells, laborious reconstitution of complicated networks would be difficult and the disparities of protein behaviors in the artificial cells have been observed. Herein, we prefer to construct artificial cells by preserving certain endogenous machinery already existing in living cells. Such principle is conducible for maintaining essential features of living cells while leaving design space for synthetic biologists to study specific cellular behavior in bottom-up manner. [5] Cell-free system, also referred to as in vitro transcription and translation system is a universal toolbox to study the organisms of cell biology. [6] Cell-free extracts inherit most of the endogenous machinery including functional macromolecules as well as metabolic networks and the addition of desired resources could initiate coupled protein expressions. Indeed, the first appearance of cell-free systems could date back to 1961 when Nirenberg and Matthaei deciphered the genetic codes. [7] Featured by the accessible utilization of molecular machinery, cellfree systems could circumvent the frangibility and membrane barrier of biological cells. The transformative development of protein synthesis using recombinant elements (PURE) system with well-defined components greatly enhances the systemic manipulation and controllability. [8] In principle, the quintessence behind cell-free system is the central dogma that reflects the flow of genetic information, from plasmid DNA to messenger RNA (mRNA), to eventually make functional proteins. As such, artificial cells encapsulated with cell-free system could serve as the valuable candidate to construct numerous biological processes. [9] The powerful ability of protein expressions, which is deprived in other synthetic systems, offers advantageous scenario for enzymatic processes that are also guided by flexible and programmable regulations of gene circuit. [10] The execution of larger genetic programs could further permit the construction of complex biochemical systems. [11] More importantly, Artificial cells that mimic the architectural and functional characteristics of living cells not only shed light on the physical principle of life but also facilitate development in the areas such as cell engineering and biomedicine. Cell-free systems carry out the central dogma that refers to the transcription and translation ...

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“…Biological systems are tightly regulated by a multifactorial interplay of biomolecular entities at both inter and intracellular scales together with extracellular environments (1,2). These entities including genes, transcripts, proteins, and metabolites, interact to function over a wide range of spatiotemporal scales (1,3) in well-orchestrated regulatory pathways (4,5). These pathways are further interlinked and form biomolecular interaction networks such as gene-regulatory, protein-protein interaction networks, and metabolic networks (6).…”

Section: Introductionmentioning

confidence: 99%

TISON: a next-generation multi-scale modeling theatre forin silicosystems oncology

Gondal

Arif

et al. 2021

Preprint

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Multi-scale models integrating biomolecular data from genetic, transcriptional, and translational levels, coupled with extracellular microenvironments can assist in decoding the complex mechanisms underlying system-level diseases such as cancer. To investigate the emergent properties and clinical translation of such cancer models, we present Theatre for in silico Systems Oncology (TISON, https://tison.lums.edu.pk), a next-generation web-based multi-scale modeling and simulation platform for in silico systems oncology. TISON provides a “zero-code” environment for multi-scale model development by seamlessly coupling scale-specific information from biomolecular networks, microenvironments, cell decision circuits, in silico cell lines, and organoid geometries. To compute the temporal evolution of multi-scale models, a simulation engine and data analysis features are also provided. Furthermore, TISON integrates patient-specific gene expression data to evaluate patient-centric models towards personalized therapeutics. Several literature-based case studies have been developed to exemplify and validate TISON’s modeling and analysis capabilities. TISON provides a cutting-edge multi-scale modeling pipeline for scale-specific as well as integrative systems oncology that can assist in drug target discovery, repositioning, and development of personalized therapeutics.

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Regulation of spatiotemporal patterning in artificial cells by a defined protein expression system

Abstract: Spatiotemporal patterning regulation in artificial cells by synthesizing a bacterial spatiotemporal regulator using a defined protein expression system.

Cited by 38 publications

References 38 publications

A Relationship between NTP and Cell Extract Concentration for Cell-Free Protein Expression

A Relationship between NTP and Cell Extract Concentration for Cell-Free Protein Expression

Emerging Advances of Cell‐Free Systems toward Artificial Cells

TISON: a next-generation multi-scale modeling theatre forin silicosystems oncology

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