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Nat Commun. 2016 Jul 29;7:12266. doi: 10.1038/ncomms12266.

Rapid construction of metabolite biosensors using domain-insertion profiling.

Author information

1
Department of Molecular &Cell Biology, University of California, Berkeley, California 94720, USA.
2
Department of Chemistry, University of California, Berkeley, California 94720, USA.
3
Energy Biosciences Institute, University of California, Berkeley, California 94720, USA.

Abstract

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.

PMID:
27470466
PMCID:
PMC4974565
DOI:
10.1038/ncomms12266
[Indexed for MEDLINE]
Free PMC Article

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