Journal: bioRxiv

Article Title: An ultra-low background far-red light-responsive optogenetic tool based on an engineered biliverdin-binding domain

doi: 10.1101/2025.11.29.691301

Figure Lengend Snippet: Fluorescence and photoswitchability of Fenix R0 and miRFP670nano (crude protein extracts from E. coli ). The OFF-state was produced with 660 nm illumination, the ON-state was produced with 530 nm illumination. Fluorescence of both states was detected at 670 nm with excitation at 647 nm. n = 3 technical replicates, mean ± sd.

Article Snippet: The gene encoding miRFP670nano (a gift from Vladislav Verkhusha; Addgene plasmid #127427) and miRFP670nano3 (Addgene plasmid #184663) with an N-terminal 6x His tag was cloned into the pBAD/HisB vector (a kind gift from Robert E. Campbell).

Techniques: Fluorescence, Produced

Journal: bioRxiv

Article Title: An ultra-low background far-red light-responsive optogenetic tool based on an engineered biliverdin-binding domain

doi: 10.1101/2025.11.29.691301

Figure Lengend Snippet: a, The directed evolution workflow for FenixS. Starting from the template Fenix R0, the full-length gene (red) was randomly mutated by error-prone PCR or site-saturation mutagenesis. The resulting library was ligated into a plasmid containing heme-oxygenase (green) and the product was used to transform E. coli . Fluorescence images of the plates after red-light (660 nm) illumination (OFF-state, red channel) and green-light (530 nm) illumination (ON-state, green channel) were overlayed. Bright (high F ON ) and red (large F ON /F OFF ) colonies were picked and cultured. Winners of each round were selected based on their brightness (F ON ), fold-change (F ON /F OFF ), and reversibility after 2 cycles (measured using crude protein extracts). Winners were used as the templates for the next round of evolution. b, Fluorescence of the ON and OFF states of selected variants after each round of directed evolution. The OFF-state was produced with 660 nm illumination, The ON-state was produced with 530 nm illumination. Fluorescence of both states was detected at 670 nm with excitation at 647 nm. Measurements were performed using crude protein extracts from E. coli . n = 3 biological replicates, mean ± sd. c, Lineage of FenixS variants starting from miRFP670nano. d, Assembly rates of apoproteins Amg2, miRFP670nano, and FenixS with BV (ratio 1.5 to 1) in vitro . e, UV-Vis absorbance spectra of the FenixS protein in its Pr (dark)-state and Pb (photo)-state. The Pb-state was obtained by irradiation for 4 minutes with 700 nm light (14 mW cm -2 ). f, Size-exclusion chromatography of FenixS at a concentration of 50 µM. Molecular weight standards are indicated.

Article Snippet: The gene encoding miRFP670nano (a gift from Vladislav Verkhusha; Addgene plasmid #127427) and miRFP670nano3 (Addgene plasmid #184663) with an N-terminal 6x His tag was cloned into the pBAD/HisB vector (a kind gift from Robert E. Campbell).

Techniques: Mutagenesis, Plasmid Preparation, Fluorescence, Cell Culture, Produced, In Vitro, Irradiation, Size-exclusion Chromatography, Concentration Assay, Molecular Weight