Dataset Information

Algorithms for the selection of fluorescent reporters.

ABSTRACT: Molecular biologists rely on the use of fluorescent probes to take measurements of their model systems. These fluorophores fall into various classes (e.g. fluorescent dyes, fluorescent proteins, etc.), but they all share some general properties (such as excitation and emission spectra, brightness) and require similar equipment for data acquisition. Selecting an ideal set of fluorophores for a particular measurement technology or vice versa is a multidimensional problem that is difficult to solve with ad hoc methods due to the enormous solution space of possible fluorophore panels. Choosing sub-optimal fluorophore panels can result in unreliable or erroneous measurements of biochemical properties in model systems. Here, we describe a set of algorithms, implemented in an open-source software tool, for solving these problems efficiently to arrive at fluorophore panels optimized for maximal signal and minimal bleed-through.

SUBMITTER: Vaidyanathan P

PROVIDER: S-EPMC7838271 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Algorithms for the selection of fluorescent reporters.

Vaidyanathan Prashant P Appleton Evan E Tran David D Vahid Alexander A Church George G Densmore Douglas D

Communications biology 20210126 1

Molecular biologists rely on the use of fluorescent probes to take measurements of their model systems. These fluorophores fall into various classes (e.g. fluorescent dyes, fluorescent proteins, etc.), but they all share some general properties (such as excitation and emission spectra, brightness) and require similar equipment for data acquisition. Selecting an ideal set of fluorophores for a particular measurement technology or vice versa is a multidimensional problem that is difficult to solve ...[more]

PMID: 33500520

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Project description:Fluorescent reporter proteins based on flavin-binding photosensors were recently developed as a new class of genetically encoded probes characterized by small size and oxygen-independent maturation of fluorescence. Flavin-based fluorescent proteins (FbFPs) address two major limitations associated with existing fluorescent reporters derived from the green fluorescent protein (GFP)-namely, the overall large size and oxygen-dependent maturation of fluorescence of GFP. However, FbFPs are at a nascent stage of development and have been utilized in only a handful of biological studies. Importantly, a full understanding of the performance and properties of FbFPs as a practical set of biological probes is lacking. In this work, we extensively characterize three FbFPs isolated from Pseudomonas putida, Bacillus subtilis, and Arabidopsis thaliana, using in vitro studies to assess probe brightness, oligomeric state, maturation time, fraction of fluorescent holoprotein, pH tolerance, redox sensitivity, and thermal stability. Furthermore, we validate FbFPs as stable molecular tags using in vivo studies by constructing a series of FbFP-based transcriptional constructs to probe promoter activity in Escherichia coli. Overall, FbFPs show key advantages as broad-spectrum biological reporters including robust pH tolerance (4-11), thermal stability (up to 60°C), and rapid maturation of fluorescence (<3 min.). In addition, the FbFP derived from Arabidopsis thaliana (iLOV) emerged as a stable and nonperturbative reporter of promoter activity in Escherichia coli. Our results demonstrate that FbFP-based reporters have the potential to address key limitations associated with the use of GFP, such as pH-sensitive fluorescence and slow kinetics of fluorescence maturation (10-40 minutes for half maximal fluorescence recovery). From this view, FbFPs represent a useful new addition to the fluorescent reporter protein palette, and our results constitute an important framework to enable researchers to implement and further engineer improved FbFP-based reporters with enhanced brightness and tighter flavin binding, which will maximize their potential benefits.

Dataset Information

Algorithms for the selection of fluorescent reporters.

Publications

Algorithms for the selection of fluorescent reporters.

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