{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,21]],"date-time":"2026-05-21T04:19:44Z","timestamp":1779337184723,"version":"3.51.4"},"reference-count":35,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2019,12,25]],"date-time":"2019-12-25T00:00:00Z","timestamp":1577232000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Sensors"],"abstract":"<jats:p>Isoleucine is one of the branched chain amino acids that plays a major role in the energy metabolism of human beings and animals. However, detailed investigation of specific receptors for isoleucine has not been carried out because of the non-availability of a tool that can monitor the metabolic flux of this amino acid in live cells. This study presents a novel genetically-encoded nanosensor for real-time monitoring of isoleucine in living cells. This nanosensor was developed by sandwiching a periplasmic binding protein (LivJ) of E. coli between a fluorescent protein pair, ECFP (Enhanced Cyan Fluorescent Protein), and Venus. The sensor, named GEII (Genetically Encoded Isoleucine Indicator), was pH stable, isoleucine-specific, and had a binding affinity (Kd) of 63 \u00b1 6 \u03bcM. The GEII successfully performed real-time monitoring of isoleucine in bacterial and yeast cells, thereby, establishing its bio-compatibility in monitoring isoleucine in living cells. As a further enhancement, in silico random mutagenesis was carried out to identify a set of viable mutations, which were subsequently experimentally verified to create a library of affinity mutants with a significantly expanded operating range (96 nM\u20131493 \u03bcM). In addition to its applicability in understanding the underlying functions of receptors of isoleucine in metabolic regulation, the GEII can also be used for metabolic engineering of bacteria for enhanced production of isoleucine in animal feed industries.<\/jats:p>","DOI":"10.3390\/s20010146","type":"journal-article","created":{"date-parts":[[2019,12,25]],"date-time":"2019-12-25T11:07:48Z","timestamp":1577272068000},"page":"146","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Real-Time Optical Detection of Isoleucine in Living Cells through a Genetically-Encoded Nanosensor"],"prefix":"10.3390","volume":"20","author":[{"given":"Shruti","family":"Singh","sequence":"first","affiliation":[{"name":"Department of Botany, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi 110062, India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maheshwar Prasad","family":"Sharma","sequence":"additional","affiliation":[{"name":"Department of Botany, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi 110062, India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Abdulaziz A.","family":"Alqarawi","sequence":"additional","affiliation":[{"name":"Plant Production Department, College of Food and Agricultural Sciences, King Saud University, P.O. Box 2460, Riyadh 11451, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Abeer","family":"Hashem","sequence":"additional","affiliation":[{"name":"Botany and Microbiology Department, College of Science, King Saud University, P.O. Box. 2460, Riyadh 11451, Saudi Arabia"},{"name":"Mycology and Plant Disease Survey Department, plant pathology Research Institute, ARC, Gaza 12511, Egypt"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8509-8953","authenticated-orcid":false,"given":"Elsayed Fathi","family":"Abd_Allah","sequence":"additional","affiliation":[{"name":"Plant Production Department, College of Food and Agricultural Sciences, King Saud University, P.O. Box 2460, Riyadh 11451, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4099-7338","authenticated-orcid":false,"given":"Altaf","family":"Ahmad","sequence":"additional","affiliation":[{"name":"Department of Botany, Aligarh Muslim University, Aligarh 202002, India"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2019,12,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"E1683","DOI":"10.1152\/ajpendo.00609.2006","article-title":"Hypoglycemic effect of isoleucine involves increased muscle glucose uptake and whole body glucose oxidation and decreased hepatic gluconeogenesis","volume":"292","author":"Doi","year":"2007","journal-title":"Am. J. Physiol. Endocrinol. 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