Cell-free biosensors for rapid detection of water contaminants

Jung, Jaeyoung K.; Alam, Khalid K.; Verosloff, Matthew S.; Capdevila, Daiana A.; Desmau, Morgane; Clauer, Phillip R.; Lee, Jeong Wook; Nguyen, Peter Q.; Pastén, Pablo; Matiasek, S. J.; Gaillard, Jean François; Giedroc, David P.; Collins, James J.; Lucks, Julius B.

doi:10.1038/s41587-020-0571-7

Cited by 285 publications

(333 citation statements)

References 51 publications

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“…The use of a fluorogenic RNA aptamer facilitated SENSR development by enabling fast and straightforward signal generation. Compared with conventional fluorescent protein outputs, the use of RNA aptamers as reporters can reduce the time it takes to observe the signal 37 .…”

Section: Discussionmentioning

confidence: 99%

“…SENSR can be implemented in any diagnostic laboratory with commonly available instruments such as a water bath and a spectrophotometer. In addition, the full potential of SENSR would be realized through the development of field-deployable devices for isothermal incubation and fluorescence measurement 37,[40][41][42][43] . Due to the simplicity of the operating mechanism, SENSR can be further developed into a portable, easy-to-use test of paper-based or lateral-flow type, as exemplified by recent developments in nucleic acid diagnostics 9,[44][45][46][47] .…”

Section: Discussionmentioning

confidence: 99%

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Sensitive fluorescence detection of SARS-CoV-2 RNA in clinical samples via one-pot isothermal ligation and transcription

et al. 2020

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The control of viral outbreaks requires nucleic acid diagnostic tests that are sensitive, simple and fast. Here, we report a highly sensitive and specific one-pot assay for the fluorescence-based detection of RNA from pathogens. The assay, which can be performed within 30-50 min of incubation time and can reach a limit of detection of 0.1-attomolar RNA concentration, relies on a sustained isothermal reaction cascade producing an RNA aptamer that binds to a fluorogenic dye. The RNA aptamer is transcribed by the T7 RNA polymerase from the ligation product of a promoter DNA probe and a reporter DNA probe that hybridize with the target single-stranded RNA sequence via the SplintR ligase (a Chlorella virus DNA ligase). In 40 nasopharyngeal SARS-CoV-2 samples, the assay reached positive and negative predictive values of 95 and 100%, respectively. We also show that the assay can rapidly detect a range of viral and bacterial RNAs.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Sensitive fluorescence detection of SARS-CoV-2 RNA in clinical samples via one-pot isothermal ligation and transcription

et al. 2020

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“…A blue light hand illuminator was designed and printed using a 3D printer to visualize the TXTL reaction output. The circuit of the illuminator, based on a modified version of Lucks et al [20], consists of eight 470nm light emitting diodes (LEDs) connected in series to two resistors: one normal and one variable. The normal resistor is used to limit the current that flows into the LEDs, and the variable resistor is used to adjust their brightness.…”

Section: Resultsmentioning

confidence: 99%

SARS-CoV-2 detection with de novo designed synthetic riboregulators

Köksaldı

Ahan

Köse

et al. 2020

Preprint

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Sars-CoV-2 is a human pathogen and is the main cause of COVID-19 disease. COVID-19 is announced as a global pandemic by World Health Organization. COVID-19 is characterized by severe conditions and early diagnosis can make dramatic changes both for personal and public health. In order to increase the reach for low cost equipment which requires a very limited technical knowledge can be beneficial to diagnose the viral infection. Such diagnostic capabilities can have a very critical role to control the transmission of the disease. Here we are reporting a state-of-the-art diagnostic tool developed by using an in vitro synthetic biology approach by employing engineered de novo riboregulators. Our design coupled with a home-made point-of-care device setting can detect and report presence of Sars-CoV-2 specific genes. The presence of Sars-CoV-2 related genes triggers translation of sfGFP mRNAs, resulting in green fluorescence output. The approach proposed here has the potential of being a game changer in Sars-COV-2 diagnostics by providing an easy-to-run, low-cost-demanding diagnostic capability.

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“…Optimized E. coli-based CFE reactions: (i) quickly synthesize grams of protein per liter in batch reactions (30)(31)(32), (ii) are scalable from the nL to 100 L scale (33,34), and (iii) can be freeze-dried for months of shelf-stability and distribution to the point of care (6,22,29,(35)(36)(37)(38)(39)(40). Freeze-dried CFE systems are poised to make disruptive impacts in biotechnology, having already been leveraged for point-of-use biosensing (41)(42)(43)(44)(45)(46), therapeutic and vaccine production (37,38,47), and educational kits (22,(48)(49)(50).…”

Section: Main Text Introductionmentioning

confidence: 99%

Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

Hershewe

Warfel

Iyer

et al. 2020

Preprint

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Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for accelerating the design of cellular function, on-demand biomanufacturing, portable diagnostics, and educational kits. Many essential biological processes that could endow CFE systems with desired functions, such as protein glycosylation, rely on the activity of membrane-bound components. However, without the use of synthetic membrane mimics, activating membrane-dependent functionality in bacterial CFE systems remains largely unstudied. Here, we address this gap by characterizing native, cell-derived membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. We first use nanocharacterization techniques to show that lipid vesicles in CFE extracts are tens to hundreds of nanometers across, and on the order of ~3x1012 particles/mL. We then determine how extract processing methods, such as post-lysis centrifugation, can be used to modulate concentrations of membrane vesicles in CFE systems. By tuning these methods, we show that increasing the number of vesicle particles to ~7x1012 particles/mL can be used to increase concentrations of heterologous membrane protein cargo expressed prior to lysis. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving N-linked and O-linked glycoprotein synthesis. We anticipate that our findings will facilitate in vitro gene expression systems that require membrane-dependent activities and open new opportunities in glycoengineering.

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Cell-free biosensors for rapid detection of water contaminants

Cited by 285 publications

References 51 publications

Sensitive fluorescence detection of SARS-CoV-2 RNA in clinical samples via one-pot isothermal ligation and transcription

Sensitive fluorescence detection of SARS-CoV-2 RNA in clinical samples via one-pot isothermal ligation and transcription

SARS-CoV-2 detection with de novo designed synthetic riboregulators

Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

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