Browse
Submit Data
Databases
API
Help

Dataset Information

31 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The design and application of genetically encodable biosensors based on fluorescent proteins.

ABSTRACT: To track the activity of cellular signaling molecules within the endogenous cellular environment, researchers have developed a diverse set of genetically encodable fluorescent biosensors. These sensors, which can be targeted to specific subcellular regions to monitor specific pools of a given signaling molecule in real time, rely upon conformational changes in a sensor domain to alter the photophysical properties of green fluorescent protein (GFP) family members. In this introductory chapter, we first discuss the properties of GFP family members before turning our attention to the design and application of genetically encodable fluorescent biosensors to live cell imaging.

SUBMITTER: Newman RH

PROVIDER: S-EPMC4109810 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

The design and application of genetically encodable biosensors based on fluorescent proteins.

Newman Robert H RH Zhang Jin J

Methods in molecular biology (Clifton, N.J.) 20140101

To track the activity of cellular signaling molecules within the endogenous cellular environment, researchers have developed a diverse set of genetically encodable fluorescent biosensors. These sensors, which can be targeted to specific subcellular regions to monitor specific pools of a given signaling molecule in real time, rely upon conformational changes in a sensor domain to alter the photophysical properties of green fluorescent protein (GFP) family members. In this introductory chapter, we ...[more]

PMID: 24052376

Similar Datasets

Genetically Encodable Fluorescent and Bioluminescent Biosensors Light Up Signaling Networks.

Project description:Cell signaling networks are intricately regulated in time and space to determine the responses and fates of cells to different cues. Genetically encodable fluorescent and bioluminescent biosensors enable the direct visualization of these spatiotemporal signaling dynamics within the native biological context, and have therefore become powerful molecular tools whose unique benefits are being used to address challenging biological questions. We first review the basis of biosensor design and remark on recent technologies that are accelerating biosensor development. We then discuss a few of the latest advances in the development and application of genetically encodable fluorescent and bioluminescent biosensors that have led to scientific or technological breakthroughs.

| S-EPMC7502535 | biostudies-literature

Genetically encoded biosensors based on engineered fluorescent proteins.

Project description:Fluorescent proteins have revolutionized cell biology by allowing researchers to non-invasively peer into the inner workings of cells and organisms. While the most common applications of fluorescent proteins are to image expression, localization, and dynamics of protein chimeras, there is a growing interest in using fluorescent proteins to create biosensors for minimally invasive imaging of concentrations of ions and small molecules, the activity of enzymes, and changes in the conformation of proteins in living cells. This tutorial review provides an overview of the progress made in the development of fluorescent protein-based biosensors to date.

| S-EPMC3000468 | biostudies-literature

Recurrent RNA motifs as scaffolds for genetically encodable small-molecule biosensors.

Project description:Allosteric RNA devices are increasingly being viewed as important tools capable of monitoring enzyme evolution, optimizing engineered metabolic pathways, facilitating gene discovery and regulators of nucleic acid-based therapeutics. A key bottleneck in the development of these platforms is the availability of small-molecule-binding RNA aptamers that robustly function in the cellular environment. Although aptamers can be raised against nearly any desired target through in vitro selection, many cannot easily be integrated into devices or do not reliably function in a cellular context. Here, we describe a new approach using secondary- and tertiary-structural scaffolds derived from biologically active riboswitches and small ribozymes. When applied to the neurotransmitter precursors 5-hydroxytryptophan and 3,4-dihydroxyphenylalanine, this approach yielded easily identifiable and characterizable aptamers predisposed for coupling to readout domains to allow engineering of nucleic acid-sensory devices that function in vitro and in the cellular context.

| S-EPMC5310984 | biostudies-literature

Development of a genetically encodable FRET system using fluorescent RNA aptamers.

Project description:Fluorescent RNA aptamers are useful as markers for tracking RNA molecules inside cells and for creating biosensor devices. Förster resonance energy transfer (FRET) based on fluorescent proteins has been used to detect conformational changes, however, such FRET devices have not yet been produced using fluorescent RNA aptamers. Here we develop an RNA aptamer-based FRET (apta-FRET) system using single-stranded RNA origami scaffolds. To obtain FRET, the fluorescent aptamers Spinach and Mango are placed in close proximity on the RNA scaffolds and a new fluorophore is synthesized to increase spectral overlap. RNA devices that respond to conformational changes are developed, and finally, apta-FRET constructs are expressed in E. coli where FRET is observed, demonstrating that the apta-FRET system is genetically encodable and that the RNA nanostructures fold correctly in bacteria. We anticipate that the RNA apta-FRET system could have applications as ratiometric sensors for real-time studies in cell and synthetic biology.

| S-EPMC5750238 | biostudies-literature

Engineering of a genetically encodable fluorescent voltage sensor exploiting fast Ci-VSP voltage-sensing movements.

Project description:Ci-VSP contains a voltage-sensing domain (VSD) homologous to that of voltage-gated potassium channels. Using charge displacement ('gating' current) measurements we show that voltage-sensing movements of this VSD can occur within 1 ms in mammalian membranes. Our analysis lead to development of a genetically encodable fluorescent protein voltage sensor (VSFP) in which the fast, voltage-dependent conformational changes of the Ci-VSP voltage sensor are transduced to similarly fast fluorescence read-outs.

| S-EPMC2429971 | biostudies-literature

Design and Prototyping of Genetically Encoded Arsenic Biosensors Based on Transcriptional Regulator AfArsR.

Project description:Genetically encoded biosensors based on engineered fluorescent proteins (FPs) are essential tools for monitoring the dynamics of specific ions and molecules in biological systems. Arsenic ion in the +3 oxidation state (As3+) is highly toxic to cells due to its ability to bind to protein thiol groups, leading to inhibition of protein function, disruption of protein-protein interactions, and eventually to cell death. A genetically encoded biosensor for the detection of As3+ could potentially facilitate the investigation of such toxicity both in vitro and in vivo. Here, we designed and developed two prototype genetically encoded arsenic biosensors (GEARs), based on a bacterial As3+ responsive transcriptional factor AfArsR from Acidithiobacillus ferrooxidans. We constructed FRET-based GEAR biosensors by insertion of AfArsR between FP acceptor/donor FRET pairs. We further designed and engineered single FP-based GEAR biosensors by insertion of AfArsR into GFP. These constructs represent prototypes for a new family of biosensors based on the ArsR transcriptional factor scaffold. Further improvements of the GEAR biosensor family could lead to variants with suitable performance for detection of As3+ in various biological and environmental systems.

| S-EPMC8470949 | biostudies-literature

Genetically encodable bioluminescent system from fungi.

Project description:Bioluminescence is found across the entire tree of life, conferring a spectacular set of visually oriented functions from attracting mates to scaring off predators. Half a dozen different luciferins, molecules that emit light when enzymatically oxidized, are known. However, just one biochemical pathway for luciferin biosynthesis has been described in full, which is found only in bacteria. Here, we report identification of the fungal luciferase and three other key enzymes that together form the biosynthetic cycle of the fungal luciferin from caffeic acid, a simple and widespread metabolite. Introduction of the identified genes into the genome of the yeast Pichia pastoris along with caffeic acid biosynthesis genes resulted in a strain that is autoluminescent in standard media. We analyzed evolution of the enzymes of the luciferin biosynthesis cycle and found that fungal bioluminescence emerged through a series of events that included two independent gene duplications. The retention of the duplicated enzymes of the luciferin pathway in nonluminescent fungi shows that the gene duplication was followed by functional sequence divergence of enzymes of at least one gene in the biosynthetic pathway and suggests that the evolution of fungal bioluminescence proceeded through several closely related stepping stone nonluminescent biochemical reactions with adaptive roles. The availability of a complete eukaryotic luciferin biosynthesis pathway provides several applications in biomedicine and bioengineering.

| S-EPMC6294908 | biostudies-literature

Red fluorescent proteins (RFPs) and RFP-based biosensors for neuronal imaging applications.

Project description:The inherent advantages of red-shifted fluorescent proteins and fluorescent protein-based biosensors for the study of signaling processes in neurons and other tissues have motivated the development of a plethora of new tools. Relative to green fluorescent proteins (GFPs) and other blue-shifted alternatives, red fluorescent proteins (RFPs) provide the inherent advantages of lower phototoxicity, lower autofluorescence, and deeper tissue penetration associated with longer wavelength excitation light. All other factors being the same, the multiple benefits of using RFPs make these tools seemingly ideal candidates for use in neurons and, ultimately, the brain. However, for many applications, the practical utility of RFPs still falls short of the preferred GFPs. We present an overview of RFPs and RFP-based biosensors, with an emphasis on their reported applications in neuroscience.

| S-EPMC4478792 | biostudies-literature

Improved genetically-encoded, FlincG-type fluorescent biosensors for neural cGMP imaging.

Project description:Genetically-encoded biosensors are powerful tools for understanding cellular signal transduction mechanisms. In aiming to investigate cGMP signaling in neurones using the EGFP-based fluorescent biosensor, FlincG (fluorescent indicator for cGMP), we encountered weak or non-existent fluorescence after attempted transfection with plasmid DNA, even in HEK293T cells. Adenoviral infection of HEK293T cells with FlincG, however, had previously proved successful. Both constructs were found to harbor a mutation in the EGFP domain and had a tail of 17 amino acids at the C-terminus that differed from the published sequence. These discrepancies were systematically examined, together with mutations found beneficial for the related GCaMP family of Ca(2+) biosensors, in a HEK293T cell line stably expressing both nitric oxide (NO)-activated guanylyl cyclase and phosphodiesterase-5. Restoring the mutated amino acid improved basal fluorescence whereas additional restoration of the correct C-terminal tail resulted in poor cGMP sensing as assessed by superfusion of either 8-bromo-cGMP or NO. Ultimately, two improved FlincGs were identified: one (FlincG2) had the divergent tail and gave moderate basal fluorescence and cGMP response amplitude and the other (FlincG3) had the correct tail, a GCaMP-like mutation in the EGFP region and an N-terminal tag, and was superior in both respects. All variants tested were strongly influenced by pH over the physiological range, in common with other EGFP-based biosensors. Purified FlincG3 protein exhibited a lower cGMP affinity (0.89 ?M) than reported for the original FlincG (0.17 ?M) but retained rapid kinetics and a 230-fold selectivity over cAMP. Successful expression of FlincG2 or FlincG3 in differentiated N1E-115 neuroblastoma cells and in primary cultures of hippocampal and dorsal root ganglion cells commends them for real-time imaging of cGMP dynamics in neural (and other) cells, and in their subcellular specializations.

| S-EPMC3781335 | biostudies-literature

Genetically Encoded Fluorescent Biosensors Illuminate the Spatiotemporal Regulation of Signaling Networks.

Project description:Cellular signaling networks are the foundation which determines the fate and function of cells as they respond to various cues and stimuli. The discovery of fluorescent proteins over 25 years ago enabled the development of a diverse array of genetically encodable fluorescent biosensors that are capable of measuring the spatiotemporal dynamics of signal transduction pathways in live cells. In an effort to encapsulate the breadth over which fluorescent biosensors have expanded, we endeavored to assemble a comprehensive list of published engineered biosensors, and we discuss many of the molecular designs utilized in their development. Then, we review how the high temporal and spatial resolution afforded by fluorescent biosensors has aided our understanding of the spatiotemporal regulation of signaling networks at the cellular and subcellular level. Finally, we highlight some emerging areas of research in both biosensor design and applications that are on the forefront of biosensor development.

| S-EPMC7462118 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data