Advances in Cell‐Free Biosensors: Principle, Mechanism, and Applications

Zhang, Liyuan; Guo, Wei; Lu, Yuan

doi:10.1002/biot.202000187

Cited by 67 publications

(62 citation statements)

References 117 publications

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“…Cell-free synthetic biology is an emerging technological platform for biosensor development in recent years due to the low-cost, one-pot configuration, and ease-to-execute [9][10][11][12]. Our recent work enabled a massively parallel cell-free protein synthesis (CFPS) from single DNA molecules encapsulated in individual femtoliter droplets and has proved that every encapsulated DNA molecule can reliably trigger the CFPS reaction in the corresponding droplet [13].…”

Section: Introductionmentioning

confidence: 99%

A single-molecule counting approach for convenient and ultrasensitive measurement of restriction digest efficiencies

et al. 2020

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Restriction endonucleases play a central role in the microbial immune system against viruses and are widely used in DNA specific cleavage, which is called restriction digestion, for genetic engineering. Herein, we applied digital cell-free protein synthesis as an easy-to-use orthogonal readout means to assess the restriction digest efficiency, a new application of digital bioassays. The digital counting principle enabled an unprecedentedly sensitive trace analysis of undigested DNA at the single-molecule level in a PCR-free manner. Our approach can quantify the template DNA of much lower concentrations that cannot be detected by ensemble-based methods such as gold-standard DNA electrophoresis techniques. The sensitive and quantitative measurements revealed a considerable variation in the digest efficiency among restriction endonucleases, from less than 70% to more than 99%. Intriguingly, none of them showed truly complete digestion within reasonably long periods of reaction time. The same rationale was extended to a multiplexed assay and applicable to any DNA-degrading or genome-editing enzymes. The enzyme kinetic parameters and the flanking sequence-dependent digest efficiency can also be interrogated with the proposed digital counting method. The absolute number of residual intact DNA molecules per microliter was concluded to be at least 107, drawing attention to the residual issue of genetic materials associated with the interpretation of nucleases’ behaviors and functions in daily genetic engineering experiments.

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Section: Introductionmentioning

confidence: 99%

A single-molecule counting approach for convenient and ultrasensitive measurement of restriction digest efficiencies

et al. 2020

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“…In the last decade, cell‐free expression systems have become increasingly popular as a new sensor platform by avoiding safety concerns associated with using living cells. Cell‐free biosensors lend faster response, higher sensitivity and more tolerance to toxic samples (Silverman et al., 2020 ; Zhang et al., 2020b ). Various cell‐free biosensors have been demonstrated to detect antibiotics (Jung et al., 2020 ), pathogens (Pardee et al., 2016 ; Takahashi et al., 2018 ), toxic substances (Lopreside et al., 2019 ; Jung et al., 2020 ), etc.…”

Section: Synthetic Biology Provides Novel Strategies To Overcome Field‐deployable Limitations Of Living Sensorsmentioning

confidence: 99%

“…E. coli ‐based cell extract is the dominating cell‐free expression system at present. To meet different application needs, further work is expected to validate the use of other non‐model organism‐based cell‐free systems as alternative cell‐free sensing platforms (Zhang et al., 2020b ).…”

Section: Outlook Towards Deploying Living Sensors In the Fieldmentioning

confidence: 99%

Programming living sensors for environment, health and biomanufacturing

Wan

Saltepe

2021

Microbial Biotechnology

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Synthetic biology offers new tools and capabilities of engineering cells with desired functions for example as new biosensing platforms leveraging engineered microbes. In the last two decades, bacterial cells have been programmed to sense and respond to various input cues for versatile purposes including environmental monitoring, disease diagnosis and adaptive biomanufacturing. Despite demonstrated proof-of-concept success in the laboratory, the realworld applications of microbial sensors have been restricted due to certain technical and societal limitations. Yet, most limitations can be addressed by new technological developments in synthetic biology such as circuit design, biocontainment and machine learning. Here, we summarize the latest advances in synthetic biology and discuss how they could accelerate the development, enhance the performance and address the present limitations of microbial sensors to facilitate their use in the field. We view that programmable living sensors are promising sensing platforms to achieve sustainable, affordable and easy-to-use on-site detection in diverse settings.

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“…Therefore, they are increasingly used in the production of complex protein products with low expression rates, aggregation, toxicity, and poor solubility in vivo (Schoborg et al 2018 ). CFPS systems also have broad application prospects and have been used in the design of rapid prototyping of DNA regulatory elements, logic systems (Chappell et al 2013 ; Karim and Jewett 2016 ; Stech and Kubick 2015 ), and biosensor devices (Lin et al 2020a , b ; Pardee et al 2016 ; Zhang et al 2020 , 2019 ).…”

Section: Introductionmentioning

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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Advances in Cell‐Free Biosensors: Principle, Mechanism, and Applications

Cited by 67 publications

References 117 publications

A single-molecule counting approach for convenient and ultrasensitive measurement of restriction digest efficiencies

A single-molecule counting approach for convenient and ultrasensitive measurement of restriction digest efficiencies

Programming living sensors for environment, health and biomanufacturing

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

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