Dual-controlled optogenetic system for the rapid down-regulation of protein levels in mammalian cells

Abstract

Optogenetic switches are emerging molecular tools for studying cellular processes as they offer higher spatiotemporal and quantitative precision than classical, chemical-based switches. Light-controllable gene expression systems designed to upregulate protein expression levels meanwhile show performances superior to their chemical-based counterparts. However, systems to reduce protein levels with similar efficiency are lagging behind. Here, we present a novel two-component, blue light-responsive optogenetic OFF switch (‘Blue-OFF’), which enables a rapid and quantitative down-regulation of a protein upon illumination. Blue-OFF combines the first light responsive repressor KRAB-EL222 with the protein degradation module B-LID (blue light-inducible degradation domain) to simultaneously control gene expression and protein stability with a single wavelength. Blue-OFF thus outperforms current optogenetic systems for controlling protein levels. The system is described by a mathematical model which aids in the choice of experimental conditions such as light intensity and illumination regime to obtain the desired outcome. This approach represents an advancement of dual-controlled optogenetic systems in which multiple photosensory modules operate synergistically. As exemplified here for the control of apoptosis in mammalian cell culture, the approach opens up novel perspectives in fundamental research and applications such as tissue engineering.

COLD REGULATED 27 and 28 are targets of CONSTITUTIVELY PHOTOMORPHOGENIC 1 and negatively affect phytochrome B signalling

“…For SEAP activity measurement, the supernatant of transfected cells was transferred to 96‐well round‐bottom microtitre plates and incubated at 68°C for 1 h to inactivate endogenous phosphatases. Afterwards, 80 μl of the supernatant was transferred to a 96‐well flat‐bottom microtitre plates, and per well 100 μl SEAP buffer (20 m m homoarginine, 1 m m MgCl 2 , 21% (v/v) diethanolamine) was added (Baaske et al ., 2018; Golonka et al ., 2019). After addition of 20 μl 120 m m para‐nitrophenyl phosphate, the absorption at 405 nm was measured for 1 h using a BMG Labtech CLARIOstar or a TriStar2 S LB 942 multimode plate reader (Berthold Technologies, Bad Wildbad, Germany) (Baaske et al ., 2018; Golonka et al ., 2019).…”

“…As positive controls, known interaction pairs were transfected (PhyA/FHL; PhyB/PIF6). Exchange of media and other cell-handling was done under 522 nm safe light, to prevent inadvertent activation of the lightsensitive systems (Baaske et al, 2018;Golonka et al, 2019). For SEAP activity measurement, the supernatant of transfected cells was transferred to 96-well round-bottom microtitre plates and incubated at 68°C for 1 h to inactivate endogenous phosphatases.…”

Synthetic Switches and Regulatory Circuits in Plants

“…red light; Beyer et al, 2015; Fig. 2A) and blue light-induced (Niopek et al, 2014(Niopek et al, , 2016 nuclear import and export of transcriptional effectors; and (2) the light-mediated degradation/depletion of proteins (Bonger et al, 2014;Baaske et al, 2018;Fig. 2B).…”

“…Simultaneously, KRAB-EL222 dimerizes binding to the C120 motif, repressing transcription of the POI-B-LID. (Adapted from Baaske et al, 2018. ) addition, fusion of the aptamer to translational repressors or enhancers permits the up-or down-regulation of the translation rate of the target protein (Pillai et al, 2004;Van Etten et al, 2012;Paek et al, 2015).…”

Phosphoregulated orthogonal signal transduction in mammalian cells

“…In nature, highly complex signaling networks have evolved to regulate diverse functions in living cells. These functions include the actions of auxiliary regulators 1 , tuning of signal detection windows 2 , controlling on/off kinetics 3,4 , enabling dynamic effects based on fast negative feedback 5 , and modulating noise resistance and ultrasensitivity 6,7 . Synthetic biology aims to capitalize on these sophisticated control systems by transferring pathways from one species to another, by engineering and rerouting existing pathways, or even by designing new pathways altogether.…”