“…Proving the idea that conformational alteration in the binding domain of the construct leads to FRET. High spatio-temporal resolution can be achieved by using such FRET sensors that provides detailed knowledge of the flux rates and intracellular concentrations of the metabolites 43,45 . The in vivo response curve was obtained by adding arsenic at concentration 10 µM to the cell suspension in 96-well microtiter plates.…”
Section: Resultsmentioning
confidence: 99%
“…Showing that the FRET is the result of conformational changes in ArsR upon binding of As 3+ . Similar FRET based approach has been used earlier to study the uptake of metabolites by measuring the dynamic changes in the FRET ratio 45 .Figure 8 In vivo quantification of the nanosensor. ( a ) Confocal image of SenALiB-676n expressed in the cytosol of S .…”
Section: Resultsmentioning
confidence: 99%
“…Region of interest (ROI) was picked and background subtraction was done to prevent artifacts in the FRET signal. Ratiometric imaging of the cells were performed and 10 min video was recorded on a confocal microscope with 1.53 N.A., 63x oil immersion objective and cooled charge coupled camera 45 to visualize the changes in the dual emission intensity ratio in a single cell (Supplementary Video 2).…”
Arsenic poisoning has been a major concern that causes severe toxicological damages. Therefore, intricate and inclusive understanding of arsenic flux rates is required to ascertain the cellular concentration and establish the carcinogenetic mechanism of this toxicant at real time. The lack of sufficiently sensitive sensing systems has hampered research in this area. In this study, we constructed a fluorescent resonance energy transfer (FRET)-based nanosensor, named SenALiB (Sensor for Arsenic Linked Blackfoot disease) which contains a metalloregulatory arsenic-binding protein (ArsR) as the As
3+
sensing element inserted between the FRET pair enhanced cyan fluorescent protein (ECFP) and Venus. SenALiB takes advantage of the ratiometic FRET readout which measures arsenic with high specificity and selectivity. SenALiB offers rapid detection response, is stable to pH changes and provides highly accurate, real-time optical readout in cell-based assays. SenALiB-676n with a binding constant (
K
d
) of 0.676 × 10
−6
M is the most efficient affinity mutant and can be a versatile tool for dynamic measurement of arsenic concentration in both prokaryotes and eukaryotes
in vivo
in a non-invasive manner.
“…Proving the idea that conformational alteration in the binding domain of the construct leads to FRET. High spatio-temporal resolution can be achieved by using such FRET sensors that provides detailed knowledge of the flux rates and intracellular concentrations of the metabolites 43,45 . The in vivo response curve was obtained by adding arsenic at concentration 10 µM to the cell suspension in 96-well microtiter plates.…”
Section: Resultsmentioning
confidence: 99%
“…Showing that the FRET is the result of conformational changes in ArsR upon binding of As 3+ . Similar FRET based approach has been used earlier to study the uptake of metabolites by measuring the dynamic changes in the FRET ratio 45 .Figure 8 In vivo quantification of the nanosensor. ( a ) Confocal image of SenALiB-676n expressed in the cytosol of S .…”
Section: Resultsmentioning
confidence: 99%
“…Region of interest (ROI) was picked and background subtraction was done to prevent artifacts in the FRET signal. Ratiometric imaging of the cells were performed and 10 min video was recorded on a confocal microscope with 1.53 N.A., 63x oil immersion objective and cooled charge coupled camera 45 to visualize the changes in the dual emission intensity ratio in a single cell (Supplementary Video 2).…”
Arsenic poisoning has been a major concern that causes severe toxicological damages. Therefore, intricate and inclusive understanding of arsenic flux rates is required to ascertain the cellular concentration and establish the carcinogenetic mechanism of this toxicant at real time. The lack of sufficiently sensitive sensing systems has hampered research in this area. In this study, we constructed a fluorescent resonance energy transfer (FRET)-based nanosensor, named SenALiB (Sensor for Arsenic Linked Blackfoot disease) which contains a metalloregulatory arsenic-binding protein (ArsR) as the As
3+
sensing element inserted between the FRET pair enhanced cyan fluorescent protein (ECFP) and Venus. SenALiB takes advantage of the ratiometic FRET readout which measures arsenic with high specificity and selectivity. SenALiB offers rapid detection response, is stable to pH changes and provides highly accurate, real-time optical readout in cell-based assays. SenALiB-676n with a binding constant (
K
d
) of 0.676 × 10
−6
M is the most efficient affinity mutant and can be a versatile tool for dynamic measurement of arsenic concentration in both prokaryotes and eukaryotes
in vivo
in a non-invasive manner.
“…Recently, well optimized FRET based nanosensors have been developed for measuring Ras and Rap1 activity [41] and imaging glutamate levels in brain [42]. We have successfully constructed special FRET based nanosensors for quantification of leucine [43] and methionine [44], for in vivo monitoring of zinc concentrations [45], lysine flux [46] and glycine betaine levels [47] and vitamin B12 levels [48].…”
a b s t r a c t Nanobiotechnology has emerged inherently as an interdisciplinary field, with collaborations from researchers belonging to diverse backgrounds like molecular biology, materials science and organic chemistry. Till the current times, researchers have been able to design numerous types of nanoscale fluorescent tool kits for monitoring protein-protein interactions through real time cellular imagery in a fluorescence microscope. It is apparent that supplementing any protein of interest with a fluorescence habit traces its function and regulation within a cell. Our review therefore highlights the application of several fluorescent probes such as molecular organic dyes, quantum dots (QD) and fluorescent proteins (FPs) to determine activity state, expression and localization of proteins in live and fixed cells. The focus is on Fluorescence Resonance Energy Transfer (FRET) based nanosensors that have been developed by researchers to visualize and monitor protein dynamics and quantify metabolites of diverse nature. FRET based toolkits permit the resolution of ambiguities that arise due to the rotation of sensor molecules and flexibility of the probe. Achievements of live cell imaging and efficient spatiotemporal resolution however have been possible only with the advent of fluorescence microscopic technology, equipped with precisely sensitive automated softwares.
“…The tool is non-invasive and has excellent spatial and temporal resolution [19,20]. At present, there are many fluorescence resonance energy transfer (FRET)-based nanosensors developed for specific analytes like glutamine, histidine, vitamin B12, lysine, arginine, and maltose [20][21][22][23][24][25].…”
(+)-Catechin is an important antioxidant of green tea (Camelia sinensis (L.) O. Kuntze). Catechin is known for its positive role in anticancerous activity, extracellular matrix degradation, cell death regulation, diabetes, and other related disorders. As a result of enormous interest in and great demand for catechin, its biosynthesis using metabolic engineering has become the subject of concentrated research with the aim of enhancing (+)-catechin production. Metabolic flux is an essential concept in the practice of metabolic engineering as it helps in the identification of the regulatory element of a biosynthetic pathway. In the present study, an attempt was made to analyze the metabolic flux of the (+)-catechin biosynthesis pathway in order to decipher the regulatory element of this pathway. Firstly, a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor (FLIP-Cat, fluorescence indicator protein for (+)-catechin) was developed for real-time monitoring of (+)-catechin flux. In vitro characterization of the purified protein of the nanosensor showed that the nanosensor was pH stable and (+)-catechin specific. Its calculated Kd was 139 µM. The nanosensor also performed real-time monitoring of (+)-catechin in bacterial cells. In the second step of this study, an entire (+)-catechin biosynthesis pathway was constructed and expressed in E. coli in two sets of plasmid constructs: pET26b-PT7-rbs-PAL-PT7-rbs-4CL-PT7-rbs-CHS-PT7-rbs-CHI and pET26b-T7-rbs-F3H-PT7-rbs- DFR-PT7-rbs-LCR. The E. coli harboring the FLIP-Cat was transformed with these plasmid constructs. The metabolic flux analysis of (+)-catechin was carried out using the FLIP-Cat. The FLIP-Cat successfully monitored the flux of catechin after adding tyrosine, 4-coumaric acid, 4-coumaroyl CoA, naringenin chalcone, naringenin, dihydroquercetin, and leucocyanidin, individually, with the bacterial cells expressing the nanosensor as well as the genes of the (+)-catechin biosynthesis pathway. Dihydroflavonol reductase (DFR) was identified as the main regulatory element of the (+)-catechin biosynthesis pathway. Information about this regulatory element of the (+)-catechin biosynthesis pathway can be used for manipulating the (+)-catechin biosynthesis pathway using a metabolic engineering approach to enhance production of (+)-catechin.
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