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.