Designing and characterization of genetically encoded isoleucine and cysteine nanosensors

Abstract

In this study, two genetically encoded nanosensors: GEII (Genetically Encoded Isoleucine Indicator) and Cys-FS (Cysteine Fluorescent Sensor) were constructed for real-time monitoring of isoleucine and cysteine in living cells. Isoleucine and cysteine are physiologically important amino acids with functions ranging from redox homeostasis to promoting intestinal health. An imbalance in the concentration of these amino acids can also act as a predictive biomarker for diseases such as Maple Syrup Urine Disease (MSUD) and Alzheimer s. However, a detailed analysis of the biological function of these amino acids hasn t been possible so far due to the lack of a nano scale, measurement tool. Therefore, a cellular/sub-cellular sensor for these amino acids will help elucidate the mechanistic underpinnings of their biological function. newline newlineBesides their biological relevance, isoleucine and cysteine are commercially important as well due to their usage in feed and pharmaceutical industries. Elite bacterial strains that over-produce these amino acids can be optimized by careful analysis of the flux of the target amino acid in bacterial cells. However, such an analysis requires probing tools that have a high temporal and spatial granularity. GEII and Cys-FS satisfy these criteria on account of being fluorescence-based genetically encoded nanosensors. While frequent measurements of changes in fluorescence allow near real-time tracking, nano scale constitution of the sensors permits fine-grained measurements of the target analyte at the sub-cellular level. newline newlineGEII and Cys-FS represent significant advancement of the state-of-the-art of sensors of these amino acids as there are no previous reported instances of genetically encoded sensors with a specific response to isoleucine and cysteine. Both the sensors were extensively characterized in vitro, with respect to their response to various amino acids, metal ions and changing pH. Both GEII and Cys-FS had a specific response to isoleucine and cysteine respectively, were unaffected by me