Showing 1 - 7 of 7 results
A Single-Component Optogenetic System Allows Stringent Switch of Gene Expression in Yeast Cells.
Light is a highly attractive actuator that allows spatiotemporal control of diverse cellular activities. In this study, we developed a single-component light-switchable gene expression system for yeast cells, termed yLightOn system. The yLightOn system is independent of exogenous cofactors, and exhibits more than a 500-fold ON/OFF ratio, extremely low leakage, fast expression kinetics, and high spatial resolution. We demonstrated the usefulness of the yLightOn system in regulating cell growth and cell cycle by stringently controlling the expression of His3 and ΔN Sic1 genes, respectively. Furthermore, we engineered a bidirectional expression module that allows the simultaneous control of the expression of two genes by light. With ClpX and ClpP as the reporters, the fast, quantitative, and spatially specific degradation of ssrA-tagged protein was observed. We suggest that this single-component optogenetic system will be immensely helpful in understanding cellular gene regulatory networks and in the design of robust genetic circuits for synthetic biology.
Light-induced protein degradation in human-derived cells.
Controlling protein degradation can be a valuable tool for posttranslational regulation of protein abundance to study complex biological systems. In the present study, we designed a light-switchable degron consisting of a light oxygen voltage (LOV) domain of Avena sativa phototropin 1 (AsLOV2) and a C-terminal degron. Our results showed that the light-switchable degron could be used for rapid and specific induction of protein degradation in HEK293 cells by light in a proteasome-dependent manner. Further studies showed that the light-switchable degron could also be utilized to mediate the degradation of secreted Gaussia princeps luciferase (GLuc), demonstrating the adaptability of the light-switchable degron in different types of protein. We suggest that the light-switchable degron offers a robust tool to control protein levels and may serves as a new and significant method for gene- and cell-based therapies.
An extraordinary stringent and sensitive light-switchable gene expression system for bacterial cells.
Light-switchable gene expression systems provide transient, non-invasive and reversible means to control biological processes with high tunability and spatiotemporal resolution. In bacterial cells, a few light-regulated gene expression systems based on photoreceptors and two-component regulatory systems (TCSs) have been reported, which respond to blue, green or red light.
Synthetic dual-input mammalian genetic circuits enable tunable and stringent transcription control by chemical and light.
Programmable transcription factors can enable precise control of gene expression triggered by a chemical inducer or light. To obtain versatile transgene system with combined benefits of a chemical inducer and light inducer, we created various chimeric promoters through the assembly of different copies of the tet operator and Gal4 operator module, which simultaneously responded to a tetracycline-responsive transcription factor and a light-switchable transactivator. The activities of these chimeric promoters can be regulated by tetracycline and blue light synergistically or antagonistically. Further studies of the antagonistic genetic circuit exhibited high spatiotemporal resolution and extremely low leaky expression, which therefore could be used to spatially and stringently control the expression of highly toxic protein Diphtheria toxin A for light regulated gene therapy. When transferring plasmids engineered for the gene switch-driven expression of a firefly luciferase (Fluc) into mice, the Fluc expression levels of the treated animals directly correlated with the tetracycline and light input program. We suggest that dual-input genetic circuits using TET and light that serve as triggers to achieve expression profiles may enable the design of robust therapeutic gene circuits for gene- and cell-based therapies.
A light-switchable bidirectional expression module allowing simultaneous regulation of multiple genes.
Several light-regulated genetic circuits have been applied to spatiotemporally control transgene expression in mammalian cells. However, simultaneous regulation of multiple genes using one genetic device by light has not yet been reported. In this study, we engineered a bidirectional expression module based on LightOn system. Our data showed that both reporter genes could be regulated at defined and quantitative levels. Simultaneous regulation of four genes was further achieved in cultured cells and mice. Additionally, we successfully utilized the bidirectional expression module to monitor the expression of a suicide gene, showing potential for photodynamic gene therapy. Collectively, we provide a robust and useful tool to simultaneously control multiple genes expression by light, which will be widely used in biomedical research and biotechnology.
Fine tuning the LightOn light-switchable transgene expression system.
Spatiotemporal control of transgene expression in living cells provides new opportunities for the characterization of gene function in complex biological processes. We previously reported a synthetic, light-switchable transgene expression system called LightOn that can be used to control gene expression using blue light. In the present study, we modified the different promoter segments of the light switchable transcription factor GAVPO and the target gene, and assayed their effects on protein expression under dark or light conditions. The results showed that the LightOn system maintained its high on/off ratio under most modifications, but its induction efficiency and background gene expression level can be fine-tuned by modifying the core promoter, the UASG sequence number, the length of the spacer between UASG and the core promoter of the target protein, and the expression level of the GAVPO transcription factor. Thus, the LightOn gene expression system can be adapted to a large range of applications according to the requirements of the background and the induced gene expression.
Spatiotemporal control of gene expression by a light-switchable transgene system.
We developed a light-switchable transgene system based on a synthetic, genetically encoded light-switchable transactivator. The transactivator binds promoters upon blue-light exposure and rapidly initiates transcription of target transgenes in mammalian cells and in mice. This transgene system provides a robust and convenient way to spatiotemporally control gene expression and can be used to manipulate many biological processes in living systems with minimal perturbation.