naa a22 4500 528787128 20180924065502.0 cr unu---uuuuu 180924e20111202xx s 000 0 eng 10.3929/ethz-b-000041336 doi 10.1371/journal.pone.0028245 doi (ETHRESEARCH)oai:www.research-collection.ethz.ch:20.500.11850/41336 Engineering Genetically Encoded Nanosensors for Real-Time In Vivo Measurements of Citrate Concentrations [Elektronische Daten] [Jennifer C. Ewald, Sabrina Reich, Stephan Baumann, Wolf B. Frommer, Nicola Zamboni] PLoS ONE Open access ethresearch Citrate is an intermediate in catabolic as well as biosynthetic pathways and is an important regulatory molecule in the control of glycolysis and lipid metabolism. Mass spectrometric and NMR based metabolomics allow measuring citrate concentrations, but only with limited spatial and temporal resolution. Methods are so far lacking to monitor citrate levels in real-time in-vivo. Here, we present a series of genetically encoded citrate sensors based on Förster resonance energy transfer (FRET). We screened databases for citrate-binding proteins and tested three candidates in vitro. The citrate binding domain of the Klebsiella pneumoniae histidine sensor kinase CitA, inserted between the FRET pair Venus/CFP, yielded a sensor highly specific for citrate. We optimized the peptide linkers to achieve maximal FRET change upon citrate binding. By modifying residues in the citrate binding pocket, we were able to construct seven sensors with different affinities spanning a concentration range of three orders of magnitude without losing specificity. In a first in vivo application we show that E. coli maintains the capacity to take up glucose or acetate within seconds even after long-term starvation. Creative Commons Attribution 3.0 Unported http://creativecommons.org/licenses/by/3.0 ethresearch Ewald Jennifer C. joint author Reich Sabrina joint author Baumann Stephan joint author Frommer Wolf B. joint author Zamboni Nicola joint author PLoS ONE Lawrence, KS, USA : Public Library of Science 6 (12), p. e28245 1932-6203 http://hdl.handle.net/20.500.11850/41336 text/html WWW-Backlink auf das Repository (Open access) 1 Journal Article ethresearch ETHRESEARCH 856 40 http://hdl.handle.net/20.500.11850/41336 text/html WWW-Backlink auf das Repository (Open access) ETHRESEARCH 700 1- Ewald Jennifer C. joint author ETHRESEARCH 700 1- Reich Sabrina joint author ETHRESEARCH 700 1- Baumann Stephan joint author ETHRESEARCH 700 1- Frommer Wolf B. joint author ETHRESEARCH 700 1- Zamboni Nicola joint author ETHRESEARCH 773 0- PLoS ONE Lawrence, KS, USA : Public Library of Science 6 (12), p. e28245 1932-6203 BK010053 XK010053 XK010000 ETHRESEARCH ETHRESEARCH ETHRESEARCH Journal Article Open access