A Green-Light-Responsive System for the Control of Transgene Expression in Mammalian and Plant Cells

Abstract

The ever-increasing complexity of synthetic gene networks and applications of synthetic biology requires precise and orthogonal gene expression systems. Of particular interest are systems responsive to light as they enable the control of gene expression dynamics with unprecedented resolution in space and time. While broadly used in mammalian backgrounds, however, optogenetic approaches in plant cells are still limited due to interference of the activating light with endogenous photoreceptors. Here, we describe the development of the first synthetic light-responsive system for the targeted control of gene expression in mammalian and plant cells that responds to the green range of the light spectrum in which plant photoreceptors have minimal activity. We first engineered a system based on the light-sensitive bacterial transcription factor CarH and its cognate DNA operator sequence CarO from Thermus thermophilus to control gene expression in mammalian cells. The system was functional in various mammalian cell lines, showing high induction (up to 350-fold) along with low leakiness, as well as high reversibility. We quantitatively described the systems characteristics by the development and experimental validation of a mathematical model. Finally, we transferred the system into A. thaliana protoplasts and demonstrated gene repression in response to green light. We expect that this system will provide new opportunities in applications based on synthetic gene networks and will open up perspectives for optogenetic studies in mammalian and plant cells.

Synthetic Switches and Regulatory Circuits in Plants

“…Despite these technical and experimental constraints, the first optogenetic tools have already been successfully implemented in plants (Table 1). These include a phytochrome-based red light-inducible and a CarH-based green light-regulated expression system (Müller et al, 2014;Ochoa-Fernandez et al, 2016;Chatelle et al, 2018). The former is activated by red light and inactivated by far-red light.…”

“…Simple supplementation of ambient illumination in greenhouses with low intensities of far-red light keeps the system repressed. Irradiation with red light leads to quantitatively controlled activation of gene expression (Müller et al, 2014;Chatelle et al, 2018). The second strategy comprises the engineering of a green lightinducible bacterial photoreceptor, CarH.…”

“…The second strategy comprises the engineering of a green lightinducible bacterial photoreceptor, CarH. Use of green light as a stimulus minimizes the interference with endogenous plant photoreceptors, as this region of the sunlight spectrum normally does not produce physiologically active signaling responses of relevance (Chatelle et al, 2018).…”

“…The development of optogenetic systems lags behind in plants, mostly because of the experimental constraints posed by the unavoidable exposure to environmental light. However, optogenetic approaches in plants have been reported, including phytochrome-and CarH-based, red-and green-light-inducible expression systems (Müller et al, 2014;Ochoa-Fernandez et al, 2016;Chatelle et al, 2018). This opens novel perspectives for engineering synthetic, light-triggered circuits in plants.…”

Green, orange, red, and far-red optogenetic tools derived from cyanobacteriochromes

“…As a target we chose Am1_c0023g2, a red/green GAF domain from Acaryochloris marina, that can bind two distinct bilins -PCB or biliverdin (BV) (27 Am1_c0023g2 can be made responsive to either green, orange, red or far-red light. Optogenetic tools that respond to green light are currently limited to complex cobalamin based systems (28,29) and CcaS/CcaR or cPAC systems that have predefined functions -gene expression (30), or adenylyl cyclase activity respectively (26). In addition, small, monomeric optogenetic tools that operate with far-red light are useful since far-red absorption provides deep tissue penetration ability for use in living animals (31).…”