Design And Application Of Genetically Encoded Biosensor For Real-time Monitoring Of Cellular Active Molecules

Intracellular metabolites play a crucial role in characterizing and regulating corresponding cellular activities. Tracking intracellular metabolites in real time by traditional means was difficult until the powerful toolkit genetically encoded biosensor was developed. In the last decades, iterative improvements of these biosensors have been made for effectively monitoring metabolites such as ATP, cAMP, cGMP, NADH, ROS, sugar, carbon monoxide, nitric oxide and so on. Endogenous genetically encoded biosensors have become powerful tools in intracellular metabolites detection in single living cells, based no the adventages such as real-time monitor, live cells analysis, high-throughput analysis, high temporal-spatial resolution, minimum interfere to cell activity.2-Oxoglutarate (2OG) is a metabolite from the highly conserved Krebs cycle and not only plays a critical role in metabolism but also acts as a signaling molecule in a variety of organisms. Environmental inorganic nitrogen is reduced to ammonium by microorganisms, whose metabolic pathways involve the conversion of2OG to glutamate and glutamine. Tracking of2OG in real-time would be useful for studies on cell metabolism and signal transduction. Here, we developed a genetically encoded2OG biosensor based on fluorescent resonance energy transfer. The dynamic range of the sensors is100uM to10mM, appeared identical to the physiological range observed in E. coli. We optimized the peptide lengths of the binding domain to obtain a sensor with a maximal ratio change of0.95upon2OG binding and demonstrated the feasibility of this sensor for the visualization of metabolites both in vitro and in vivo. We also developed a novel sensor by inserting the functional2OG-binding domain GAF of the NifA protein into YFP. This sensor was found to be highly specific to2OG Following binding of2OG, fluorescence intensity of the sensor increased with increasing2OG concentration and reached a1.5-fold maximum fluorescence signal change, kinetics of fluorescence signal upon2OG association with sensor was fast, the dynamic response range of the mOGsor sensors was100uM-100mM. This sensor reported cellular2OG dynamics in E. coli cells in real time upon different nutrition challenges and manifested the differences in2OG pool accumulation and depletion velocity.Quorum sensing (QS) is a universal phenomenon that exists in various bacterial species and produces and monitors signaling molecules to regulate specific sets of genes in a population density-dependent manner. The QS system is involved in many important biological functions such as luminescence, antibiotic production, and biofilm formation. The autoinducer N-(3-oxo-hexanoyl)-L-homoserine lactone (3OC6HSL), an N-acylhomoserine lactone (AHL), plays a significant role in the QS system of the marine bacterium Vibrio fischeri. Tracing3OC6HSL would be significant in studies related to QS signal transduction. Traditional detection of QS signaling molecules has relied primarily on bacterial reporter strains and high-performance liquid chromatography, which are time consuming and have low sensitivity. Because3OC6HSL binding to LuxR from V. fischeri causes a conformational change, we developed a genetically encoded biosensor based on Forster resonance energy transfer (FRET) by inserting LuxR between the FRET pair YFP/CFP and demonstrated the feasibility of this sensor for visualizing3OC6HSL both in vitro and in vivo.