From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression

Charting the Signal Trajectory in a Light-Oxygen-Voltage Photoreceptor by Random Mutagenesis and Covariance Analysis

“…Random mutations were introduced into the LOV photosensor of YF1 including a C-terminal coiled-coil denoted J␣ (amino acids 2-146) via error-prone PCR (19 -21). Mutagenesis was performed in the background of the pDusk-DsRed reporter plasmid (18), which encodes YF1 and its cognate response regulator FixJ that drives expression of the fluorescent reporter DsRed, thus facilitating rapid screening of photoreceptor variants. Upon transformation of the resultant plasmid library into E. coli, single clones expressing individual YF1 variants were separated by plating on agar.…”

“…We generated two replica copies of the clone library, one was incubated in constant darkness, and one was incubated under constant blue light (100 W cm Ϫ2 470 nm). Under these conditions, clones harboring wild-type YF1 express the reporter DsRed to different levels depending upon light conditions, with high levels when incubated in the dark and low levels when incubated under blue light (18). DsRed expression levels can conveniently be gauged on plate by fluorescence via visual inspection through a 590-nm long-pass filter (Fig.…”

“…Upon DNA sequencing and accounting for duplicate mutations that occurred in several clones, we thus identified 42 single mutants (Table 1) and 102 YF1 variants that bore more than one mutation (supplemental Table S1). For all constructs, we measured DsRed expression and optical density at 600 nm (A 600 ) following incubation under dark and blue light conditions (25 W cm Ϫ2 470 nm) in 96-well format, which affords higher throughput than our previous setup (17,18) (Fig. 3A and supplemental Table S1).…”

“…Facilitated by the availability of efficient functional assays (18) and high-resolution structural information (17), we took a twopronged approach to identify residues that govern signal transduction: random mutagenesis for in vivo identification and large-scale sequence covariance analysis for in silico identification. We thus find numerous mutations within the LOV domain that abolish or invert the signal response to incident blue light.…”

LOV and BLUF flavoproteins’ regulatory photoreceptors of microorganisms and photosensory actuators in optogenetic systems

“…На основе свойства бактериальных LOV-доменных фоторецепторов включать в состав в качестве эффекторного домена H-киназу скон-струирована синтетическая регулируемая светом H-киназа, у которой нефоточувствительный PAS-домен от сенсора кислорода FixL из бактерий Bradyrhizobium japonicum заменен на LOV-домен фоторецептора YtvA из B. subtilis [36]. Дальнейшее применение этого гибридного белка связано с ин-женерией плазмид для светорегулируемой индукции (pDawn) или репрессии (pDusk) бактериальных генов [37].…”