scholarly journals Real-Time Optical Detection of Isoleucine in Living Cells through a Genetically-Encoded Nanosensor

Sensors ◽  
2019 ◽  
Vol 20 (1) ◽  
pp. 146
Author(s):  
Shruti Singh ◽  
Maheshwar Prasad Sharma ◽  
Abdulaziz A. Alqarawi ◽  
Abeer Hashem ◽  
Elsayed Fathi Abd_Allah ◽  
...  
Keyword(s):  
Real Time ◽  
Metabolic Regulation ◽  
Metabolic Flux ◽  
Fluorescent Protein ◽  
Animal Feed ◽  
Living Cells ◽  
Yeast Cells ◽  
Human Beings ◽  
Real Time Monitoring ◽  
Enhanced Production

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 ± 6 μM. 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–1493 μM). 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.

A multicolor DNA tetrahedron nanoprobe for analyzing human telomerase in living cells

2021 ◽  
Author(s):  
Ruiyuan Zhang ◽  
Ruixue Zhang ◽  
Wei Jiang ◽  
Xiaowen Xu
Keyword(s):  
Real Time ◽  
Length Distribution ◽  
Telomerase Activity ◽  
Living Cells ◽  
Real Time Monitoring ◽  
Dna Tetrahedron ◽  
Human Telomerase

A sequentially lighting-up multicolor DNA tetrahedron nanoprobe is constructed for imaging telomerase activity, real-time monitoring telomerase action and determining product length distribution in living cells.

A novel microfluidic microelectrode chip for a significantly enhanced monitoring of NPY-receptor activation in live mode

Lab on a Chip ◽  
2017 ◽  
Vol 17 (24) ◽  
pp. 4294-4302 ◽  
Author(s):  
Franziska D. Zitzmann ◽  
Heinz-Georg Jahnke ◽  
Felix Nitschke ◽  
Annette G. Beck-Sickinger ◽  
Bernd Abel ◽  
...  
Keyword(s):  
Real Time ◽  
Microfluidic Chip ◽  
Microelectrode Array ◽  
Fem Simulation ◽  
Receptor Activation ◽  
Living Cells ◽  
Real Time Monitoring ◽  
Non Invasive ◽  
Npy Receptor ◽  
Simulation Based

We present a FEM simulation based step-by-step development of a microelectrode array integrated into a microfluidic chip for the non-invasive real-time monitoring of living cells.

scholarly journals Ligand-Mediated Assembly and Real-Time Cellular Dynamics of Estrogen Receptor α-Coactivator Complexes in Living Cells

2001 ◽  
Vol 21 (13) ◽  
pp. 4404-4412 ◽  
Author(s):  
David L. Stenoien ◽  
Anne C. Nye ◽  
Maureen G. Mancini ◽  
Kavita Patel ◽  
Martin Dutertre ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Real Time ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Steroid Receptor ◽  
Estrogen Receptor Α ◽  
Living Cells ◽  
Live Cells ◽  
Creb Binding Protein ◽  
High Accumulation

ABSTRACT Studies with live cells demonstrate that agonist and antagonist rapidly (within minutes) modulate the subnuclear dynamics of estrogen receptor α (ER) and steroid receptor coactivator 1 (SRC-1). A functional cyan fluorescent protein (CFP)-taggedlac repressor-ER chimera (CFP-LacER) was used in live cells to discretely immobilize ER on stably integratedlac operator arrays to study recruitment of yellow fluorescent protein (YFP)-steroid receptor coactivators (YFP–SRC-1 and YFP-CREB binding protein [CBP]). In the absence of ligand, YFP–SRC-1 is found dispersed throughout the nucleoplasm, with a surprisingly high accumulation on the CFP-LacER arrays. Agonist addition results in the rapid (within minutes) recruitment of nucleoplasmic YFP–SRC-1, while antagonist additions diminish YFP–SRC-1–CFP-LacER associations. Less ligand-independent colocalization is observed with CFP-LacER and YFP-CBP, but agonist-induced recruitment occurs within minutes. The agonist-induced recruitment of coactivators requires helix 12 and critical residues in the ER–SRC-1 interaction surface, but not the F, AF-1, or DNA binding domains. Fluorescence recovery after photobleaching indicates that YFP–SRC-1, YFP-CBP, and CFP-LacER complexes undergo rapid (within seconds) molecular exchange even in the presence of an agonist. Taken together, these data suggest a dynamic view of receptor-coregulator interactions that is now amenable to real-time study in living cells.

scholarly journals Designing, construction and characterization of genetically encoded FRET-based nanosensor for real time monitoring of lysine flux in living cells

2016 ◽  
Vol 14 (1) ◽  
Author(s):  
Seema Ameen ◽  
Mohammad Ahmad ◽  
Mohd. Mohsin ◽  
M. Irfan Qureshi ◽  
Mohamed M. Ibrahim ◽  
...  
Keyword(s):  
Real Time ◽  
Living Cells ◽  
Real Time Monitoring

Real-time monitoring of endogenous cysteine levels in living cells using a CD-based ratiometric fluorescent nanoprobe

2018 ◽  
Vol 410 (18) ◽  
pp. 4379-4386 ◽  
Author(s):  
Hong Wang ◽  
Peisheng Zhang ◽  
Yong Tian ◽  
Yuan Zhang ◽  
Heping Yang ◽  
...  
Keyword(s):  
Real Time ◽  
Living Cells ◽  
Real Time Monitoring ◽  
Fluorescent Nanoprobe
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