A minimalist biosensor: Quantitation of cyclic di-GMP using the conformational change of a riboswitch aptamer

Kellenberger, Colleen A.; Sales-Lee, Jade; Pan, Yu‐Chen; Gassaway, Madalee M.; Herr, Amy E.; Hammond, Ming C.

doi:10.1080/15476286.2015.1062970

Cited by 27 publications

(13 citation statements)

References 47 publications

(77 reference statements)

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“…Advances in generating synthetic riboswitches have accelerated their application as genetic controllers in the areas of molecular sensors, metabolic pathway optimization (Cress et al, ; Dietrich et al, ; Liu et al, ; McKeague et al, ; Rogers et al, ) and therapeutic applications (Davydova et al, ; Lee et al, ). In the past decade, several natural and artificial RNA‐based sensors have been engineered to respond to a wide range of metabolites, including folinic acid (Trausch et al, ), theophylline (Beisel et al, ; Lynch et al, ; Michener and Smolke, ; Topp et al, ; Wachsmuth et al, ), xanthine (Beisel et al, ), tetracycline (Beisel et al, ; Weigand and Suess, ), ammeline (Dixon et al, ), cyclic‐di‐GMP (Kellenberger et al, ; Lynch et al, ), cyclic‐di‐AMP (Kellenberger et al, ), β‐catenin (Bloom et al, ), thiamine 5′‐pyrophosphate (TPP) (You et al, ), guanine (Paige et al, ; You et al, ), adenine (You et al, ), S‐adenosyl‐methionine (SAM) (Paige et al, ; You et al, ), adenosine 5‐diphosphate (ADP) (Paige et al, ), guanosine 5‐triphosphate (GTP) (Paige et al, ), flavin mononucleotide (FMN) (Meyer et al, ), lysine (Yang et al, ; Zhou and Zeng, ), glucosamine 6‐phosphate (Lee and Oh, ) in prokaryotic, eukaryotic, and mammalian cells through various mechanisms.…”

Section: Introductionmentioning

confidence: 99%

Naringenin‐responsive riboswitch‐based fluorescent biosensor module for Escherichia coli co‐cultures

Xiu

Jang

Jones

et al. 2017

Biotech & Bioengineering

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The ability to design and construct combinatorial synthetic metabolic pathways has far exceeded our capacity for efficient screening and selection of the resulting microbial strains. The need for high-throughput rapid screening techniques is of upmost importance for the future of synthetic biology and metabolic engineering. Here we describe the development of an RNA riboswitch-based biosensor module with dual fluorescent reporters, and demonstrate a high-throughput flow cytometry-based screening method for identification of naringenin over producing Escherichia coli strains in co-culture. Our efforts helped identify a number of key operating parameters that affect biosensor performance, including the selection of promoter and linker elements within the sensor-actuator domain, and the effect of host strain, fermentation time, and growth medium on sensor dynamic range. The resulting biosensor demonstrates a high correlation between specific fluorescence of the biosensor strain and naringenin titer produced by the second member of the synthetic co-culture system. This technique represents a novel application for synthetic microbial co-cultures and can be expanded from naringenin to any metabolite if a suitable riboswitch is identified. The co-culture technique presented here can be applied to a variety of target metabolites in combination with the SELEX approach for aptamer design. Due to the compartmentalization of the two genetic constructs responsible for production and detection into separate cells and application as independent modules of a synthetic microbial co-culture we have subsequently reduced the need for re-optimization of the producer module when the biosensor is replaced or removed. Biotechnol. Bioeng. 2017;114: 2235-2244. © 2017 Wiley Periodicals, Inc.

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Section: Introductionmentioning

confidence: 99%

Naringenin‐responsive riboswitch‐based fluorescent biosensor module for Escherichia coli co‐cultures

Xiu

Jang

Jones

et al. 2017

Biotech & Bioengineering

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“…Our results demonstrate that fluorescence‐protein‐based FRET biosensors can be employed as powerful analytical tools to detect c‐di‐GMP in the nanomolar range and to monitor performance of diguanylate cyclase enzyme reactions in a 384‐well format in the low nanomolar range. With increasing numbers of available assays that monitor fluctuations in cellular c‐di‐GMP levels, we can begin to identify effective approaches by which to discover potent small‐molecule modulators of c‐di‐GMP signaling and to monitor their phenotypic effects and influence on cellular c‐di‐GMP signaling patterns. In addition to c‐di‐GMP, many other cyclic nucleotide second messengers, such as cAMP, cGMP, c‐di‐AMP, and cGAMP, currently lack high‐throughput‐screening (HTS)‐capable analytical methods that allow in situ detection of their levels during enzymatic assays or in complex biological samples.…”

Section: Discussionmentioning

confidence: 99%

Identification of Small‐Molecule Modulators of Diguanylate Cyclase by FRET‐Based High‐Throughput Screening

et al. 2018

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The bacterial second messenger cyclic diguanosine monophosphate (c‐di‐GMP) is a key regulator of cellular motility, the cell cycle, and biofilm formation with its resultant antibiotic tolerance, which can make chronic infections difficult to treat. Therefore, diguanylate cyclases, which regulate the spatiotemporal production of c‐di‐GMP, might be attractive drug targets for control of biofilm formation that is part of chronic infections. We present a FRET‐based biochemical high‐throughput screening approach coupled with detailed structure–activity studies to identify synthetic small‐molecule modulators of the diguanylate cyclase DgcA from Caulobacter crescentus. We identified a set of seven small molecules that regulate DgcA enzymatic activity in the low‐micromolar range. Subsequent structure–activity studies on selected scaffolds revealed a remarkable diversity of modulatory behavior, including slight chemical substitutions that reverse the effects from allosteric enzyme inhibition to activation. The compounds identified represent new chemotypes and are potentially developable into chemical genetic tools for the dissection of c‐di‐GMP signaling networks and alteration of c‐di‐GMP‐associated phenotypes. In sum, our studies underline the importance of detailed mechanism‐of‐action studies for inhibitors of c‐di‐GMP signaling and demonstrate the complex interplay between synthetic small molecules and the regulatory mechanisms that control the activity of diguanylate cyclases.

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“…Our results demonstrate that fluorescence protein based FRET biosensors can be employed as powerful analytical tools to detect c-di-GMP in the nanomolar range and monitor performance of diguanylate cyclase enzyme reactions in a 384 well format in the low nanomolar range. With increasing numbers of assays available that monitor fluctuations in cellular c-di-GMP levels (10,(15)(16)(17), we can begin to identify effective approaches to discover potent small molecule modulators of c-di-GMP signaling and monitor their phenotypic effects and influence on cellular c-di-GMP signaling patterns. In addition to c-di-GMP, many other cyclic nucleotide second messengers such as cAMP, cGMP, c-di-AMP and cGAMP currently lack HTS capable analytical methods that allow in situ detection of their levels during enzymatic assays or within complex biological samples.…”

Section: Discussionmentioning

confidence: 99%

Identification of Small Molecule Modulators of Diguanylate Cyclase by FRET-based High-Throughput-Screening

Christen

Kamischke

Kulasekara

et al. 2018

Preprint

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The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) is a key regulator of cellular motility, the cell cycle, and biofilm formation with its resultant antibiotic tolerance, which may make chronic infections difficult to treat. Therefore, diguanylate cyclases, which regulate the spatiotemporal production of c-di-GMP, may be attractive drug targets to control biofilm formation that is part of chronic infections. In this paper, we present a FRET-based biochemical high-throughput screening approach coupled with detailed structure-activity studies to identify synthetic small molecule modulators of the diguanylate cyclase, DgcA, from Caulobacter crescentus. We identified a set of 7 small molecules that in the low µM range regulate DgcA enzymatic activity. Subsequent structure activity studies on selected scaffolds revealed a remarkable diversity of modulatory behaviors, including slight chemical substitutions that revert the effects from allosteric enzyme inhibition to activation. The compounds identified represent novel chemotypes and are potentially developable into chemical genetic tools for the dissection of c-di-GMP signaling networks and alteration of c-di-GMP associated phenotypes. In sum, our studies underline the importance for detailed mechanism of action studies for inhibitors of c-di-GMP signaling and demonstrate the complex interplay between synthetic small molecules and the regulatory mechanisms that control the activity of diguanylate cyclases.

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A minimalist biosensor: Quantitation of cyclic di-GMP using the conformational change of a riboswitch aptamer

Cited by 27 publications

References 47 publications

Naringenin‐responsive riboswitch‐based fluorescent biosensor module for Escherichia coli co‐cultures

Naringenin‐responsive riboswitch‐based fluorescent biosensor module for Escherichia coli co‐cultures

Identification of Small‐Molecule Modulators of Diguanylate Cyclase by FRET‐Based High‐Throughput Screening

Identification of Small Molecule Modulators of Diguanylate Cyclase by FRET-based High-Throughput-Screening

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