SDS PAGE analysis of purified FbFPs.

<p>FbFPs were purified using nickel affinity and anion exchange chromatography. A) Purified FbFPs were loaded on a denaturing polyacrylamide gel and allowed to migrate at 160 V for 40 min. Lane 1: iLOV after nickel affinity chromatography, lane 2: iLOV after anion exchange chromatography, lane 3: PpFbFP after nickel affinity chromatography, lane 4: PpFbFP after anion exchange chromatography, lane 5: EcFbFP after nickel affinity chromatography, lane 6: EcFbFP after anion exchange chromatography. B) Purified PpFbFP and iLOV emit cyan-green fluorescence upon excitation using a UV transilluminator.</p

Quantum yield and brightness of FbFPs.

<p>Brightness is quantified as the product of quantum yield and molar extinction coefficient of the fluorophore (flavin mononucleotide). Brightness values are reported for monomeric units of PpFbFP and EcFbFP, which exist as functional dimers.</p

Effect of pH on FbFP fluorescence.

<p>Histograms depict the fraction of peak fluorescence (measured at 495 nm at pH 7 for PpFbFP and EcFbFP and pH 6 for iLOV) retained by A) PpFbFP, B) EcFbFP, and C) iLOV after incubation in buffers of pH 2, 4, 10, and 11 for 2.5 h. D) FbFPs are readily denatured by incubation at pH 2 and are characterized by the appearance of a flavin-spectrum with a peak at 525 nm, as is depicted for iLOV.</p

FbFPs as transcriptional reporters of T5 promoter activity in <i>E.coli</i> grown in M9-glucose.

<p>FbFPs and YFP were expressed in <i>E. coli</i> MG1655 cells using an IPTG-inducible T5 promoter. Fluorescence and optical density were recorded over the logarithmic phase of cell growth (typically after ≈2 hours of lag phase following re-inoculation of overnight culture) in M9 medium supplemented with glucose at 20 mM concentration as the carbon source. IPTG concentrations were varied to span different levels of transcriptional activity of the T5 promoter. Fluorescence was divided by the optical density at 600 nm. Normalized fluorescence values are depicted for A) PpFbFP, B) EcFbFP, C) iLOV, and D) YFP. Steady state promoter activity was verified using YFP as a reporter. PpFbFP and EcFbFP deviated considerably from the expected steady state promoter dynamics. However, iLOV revealed close agreement with the YFP expression profile over a broad range of IPTG concentrations. Promoter activities are depicted corresponding only to the logarithmic phase of cell growth (0.4< <i>A_600nm</i> <0.8). As the duration of the logarithmic phase varies for <i>E. coli</i> cells expressing distinct transcriptional reporter constructs, so does the time frame over which the promoter activity is depicted in the figures.</p

iLOV as a reporter of phage lambda promoter activity in <i>E. </i>coli.

<p>GFP and iLOV were expressed in <i>E. coli</i> MG1655 cells using a constitutive phage lambda promoter. Fluorescence and optical density were recorded over the logarithmic phase of cell growth (typically after ≈2 hours of lag phase following re-inoculation of overnight culture) in M9 medium supplemented with glucose at 20 mM or glycerol at 0.5% as the carbon source. Fluorescence was divided by the optical density at 600 nm and normalized to the maximum value reached over 16 h. of cell growth. Steady state promoter activity was verified using GFP as a reporter. We generally observed good agreement between the iLOV and GFP expression profiles. Promoter activities are shown corresponding only to the logarithmic phase of cell growth (0.4< <i>A_600nm</i> <0.8). As the duration of the logarithmic phase varies for <i>E. coli</i> cells expressing the GFP and iLOV reporter constructs, so does the time frame over which the phage λ promoter activity is depicted in the figures.</p

Characterization of Flavin-Based Fluorescent Proteins: An Emerging Class of Fluorescent Reporters

<div><p>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 <i>Pseudomonas putida</i>, <i>Bacillus subtilis</i>, and <i>Arabidopsis thaliana,</i> using <i>in vitro</i> 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 <i>in vivo</i> studies by constructing a series of FbFP-based transcriptional constructs to probe promoter activity in <i>Escherichia coli</i>. 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 <i>Arabidopsis thaliana</i> (iLOV) emerged as a stable and nonperturbative reporter of promoter activity in <i>Escherichia coli</i>. 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.</p></div

Fraction of fluorescent holoprotein.

<p>The fraction of FbFP that exists in an FMN-bound holo form (<i>F_holo</i>) is calculated based on absorption at 450 nm by the holoprotein.</p

Expression of iLOV in anaerobically cultured <i>E. coli</i> MG1655.

<p><i>E. coli</i> MG1655 cells were grown in M9 medium supplemented with 20 mM glucose (carbon source) and 20 mM potassium nitrate (electron acceptor). Anaerobic conditions were established by growing the cells in air-tight stoppered Balch tubes filled completely with growth media and evacuated for 30 minutes using vacuum suction. Anaerobic cultures were grown without shaking. Cells were induced with 0.5 mM IPTG for 12 hours, resuspended in phosphate buffered saline and scanned using fluorescence spectrometry (470 nm excitation).</p

Effect of temperature on FbFP fluorescence.

<p>Histograms depict the fraction of peak fluorescence (measured at 495 nm at room temperature) retained by A) PpFbFP, B) EcFbFP, and C) iLOV after incubation at 40°C, 50°C, 60°C, and 70°C for 2.5 h. D) FbFPs are readily denatured at 70°C and are characterized by the appearance of a flavin-spectrum with a peak at 525 nm, as is depicted for iLOV.</p

Doubling times of <i>E. coli</i> MG1655 expressing FbFPs, GFP, and YFP.

<p>PpFbFP, EcFbFP, iLOV, and YFP were expressed in <i>E. coli</i> MG1655 using an IPTG-inducible phage T5 promoter harbored in a medium copy plasmid (pQE80L). In addition, iLOV and GFP were expressed using a constitutive phage lamda promoter in a low copy plasmid (pAM06-tet). Cells were grown in M9 media using glycerol or glucose as the carbon source and doubling times were estimated over the logarithmic phase of cell growth corresponding to 0.4< <i>A_600nm</i> <0.8.</p