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Rapid construction of metabolite biosensors using domain-insertion profiling

Single-fluorescent protein biosensors (SFPBs) are an important class of probes that enable the single-cell quantification of analytes in vivo. Despite advantages over other detection technologies, their use has been limited by the inherent challenges of their construction. Specifically, the rational...

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Autores principales: Nadler, Dana C., Morgan, Stacy-Anne, Flamholz, Avi, Kortright, Kaitlyn E., Savage, David F.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4974565/
https://www.ncbi.nlm.nih.gov/pubmed/27470466
http://dx.doi.org/10.1038/ncomms12266
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author Nadler, Dana C.
Morgan, Stacy-Anne
Flamholz, Avi
Kortright, Kaitlyn E.
Savage, David F.
author_facet Nadler, Dana C.
Morgan, Stacy-Anne
Flamholz, Avi
Kortright, Kaitlyn E.
Savage, David F.
author_sort Nadler, Dana C.
collection PubMed
description Single-fluorescent protein biosensors (SFPBs) are an important class of probes that enable the single-cell quantification of analytes in vivo. Despite advantages over other detection technologies, their use has been limited by the inherent challenges of their construction. Specifically, the rational design of green fluorescent protein (GFP) insertion into a ligand-binding domain, generating the requisite allosteric coupling, remains a rate-limiting step. Here, we describe an unbiased approach, termed domain-insertion profiling with DNA sequencing (DIP-seq), that combines the rapid creation of diverse libraries of potential SFPBs and high-throughput activity assays to identify functional biosensors. As a proof of concept, we construct an SFPB for the important regulatory sugar trehalose. DIP-seq analysis of a trehalose-binding-protein reveals allosteric hotspots for GFP insertion and results in high-dynamic range biosensors that function robustly in vivo. Taken together, DIP-seq simultaneously accelerates metabolite biosensor construction and provides a novel tool for interrogating protein allostery.
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spelling pubmed-49745652016-08-18 Rapid construction of metabolite biosensors using domain-insertion profiling Nadler, Dana C. Morgan, Stacy-Anne Flamholz, Avi Kortright, Kaitlyn E. Savage, David F. Nat Commun Article Single-fluorescent protein biosensors (SFPBs) are an important class of probes that enable the single-cell quantification of analytes in vivo. Despite advantages over other detection technologies, their use has been limited by the inherent challenges of their construction. Specifically, the rational design of green fluorescent protein (GFP) insertion into a ligand-binding domain, generating the requisite allosteric coupling, remains a rate-limiting step. Here, we describe an unbiased approach, termed domain-insertion profiling with DNA sequencing (DIP-seq), that combines the rapid creation of diverse libraries of potential SFPBs and high-throughput activity assays to identify functional biosensors. As a proof of concept, we construct an SFPB for the important regulatory sugar trehalose. DIP-seq analysis of a trehalose-binding-protein reveals allosteric hotspots for GFP insertion and results in high-dynamic range biosensors that function robustly in vivo. Taken together, DIP-seq simultaneously accelerates metabolite biosensor construction and provides a novel tool for interrogating protein allostery. Nature Publishing Group 2016-07-29 /pmc/articles/PMC4974565/ /pubmed/27470466 http://dx.doi.org/10.1038/ncomms12266 Text en Copyright © 2016, The Author(s) http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Nadler, Dana C.
Morgan, Stacy-Anne
Flamholz, Avi
Kortright, Kaitlyn E.
Savage, David F.
Rapid construction of metabolite biosensors using domain-insertion profiling
title Rapid construction of metabolite biosensors using domain-insertion profiling
title_full Rapid construction of metabolite biosensors using domain-insertion profiling
title_fullStr Rapid construction of metabolite biosensors using domain-insertion profiling
title_full_unstemmed Rapid construction of metabolite biosensors using domain-insertion profiling
title_short Rapid construction of metabolite biosensors using domain-insertion profiling
title_sort rapid construction of metabolite biosensors using domain-insertion profiling
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4974565/
https://www.ncbi.nlm.nih.gov/pubmed/27470466
http://dx.doi.org/10.1038/ncomms12266
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