Light-switchable System Research Articles

OPTO-BLUE: An Integrated Bidirectional Optogenetic Lentiviral Platform for Controlled Light-Induced Gene Expression.

Regulated systems for transgene expression are useful tools in basic research and a promising platform in biomedicine due to their regulated transgene expression by an inducer. The emergence of optogenetics expression systems enabled the construction of light-switchable systems, enhancing the spatial and temporal resolution of a transgene. The LightOn system is an optogenetic tool that regulates the expression of a gene of interest using blue light as an inducer. This system is based on a photosensitive protein (GAVPO), which dimerizes and binds to the UASG sequence in response to blue light, triggering the expression of a downstream transgene. Previously, we adapted the LightOn system to a dual lentiviral vector system for neurons. Here, we continue the optimization and assemble all components of the LightOn system into a single lentiviral plasmid, the OPTO-BLUE system. For functional validation, we used enhanced green fluorescent protein (EGFP) as an expression reporter (OPTO-BLUE-EGFP) and evaluated the efficiency of EGFP expression by transfection and transduction in HEK293-T cells exposed to continuous blue-light illumination. Altogether, these results prove that the optimized OPTO-BLUE system allows the light-controlled expression of a reporter protein according to a specific time and light intensity. Likewise, this system should provide an important molecular tool to modulate gene expression of any protein by blue light.

Arduino-based Automatic Light Switching System

This article focuses on the problem of high power consumption caused by long working hours of lights, and designs an automatic light switch system based on the number of people to achieve energy-saving effects. After experiencing the process of selecting hardware, designing circuits, writing programs, structural design, 3D modeling, 3D printing processing, assembly, etc., an automatic light on/off system has been designed, which is low-cost and can achieve automatic light on/off based on Arduino microcontroller technology, playing a role in saving electricity to a certain extent.

(Keynote) Electromagnetic Field Stimulated MOFs

Electromagnetic, external stimuli can be used to switch Metal-Organic Framework (MOF) materials and to achieve control over the pores of these materials on different ways. We designed MOFs with switchable moieties in their side-chains, such as azobenzene, to be switchable under irradiation of UV/Vis light. Additionally, we developed devices to be able to measure the impact of electromagnetic fields in-situ. We focused on surface anchored MOF (SURMOF) thin films, deposited on asymmetric, porous α-Al2O3 supports as gas separation membrane layers. It is possible to control the gas transport properties of the materials using, but also using electric fields, and measure changes gas separation (permeability and selectivity) of binary mixtures in-situ, using the Wicke-Kallenbach technique. The light switchable systems were investigated on their CO2 separation performance: By switching the azobenzene between cis and trans isomers inside the pore windows, it was possible to switch dipolar moments, thus changing the adsorption properties towards the quadrupolar moment of CO2. Using electric fields of 500 V/mm we could show on the prominent example of ZIF-8, a zeolitic imidazolate framework, that it has a strong impact on the gas transport properties, especially looking at the molecular sieving. The mechanistic of the electric field influence on the ZIF is as follows: The electric field distorts the lattice of the MOF, shifting the Zn2+ ions, lowering the symmetry of the unit cell. This effect leads to polarized polymophs of the structure, sharpening the molecular sieving through a stiffening effect. The purification of Propylene/Propane, a highly desired separation for the plastics industry, could be increased through the electric field stimuli by 33%.References Knebel, A. et al. Defibrillation of soft porous metal-organic frameworks with electric fields. Science 358, 347–351; 10.1126/science.aal2456 (2017).Wang, Z. et al. Tunable molecular separation by nanoporous membranes. Nat. Commun. 7, 13872; 10.1038/ncomms13872 (2016).Müller, K. et al. Switching Thin Films of Azobenzene-Containing Metal-Organic Frameworks with Visible Light. Chem. Eur. J. 23, 5434–5438; 10.1002/chem.201700989 (2017).Knebel, A. et al. Azobenzene Guest Molecules as Light-Switchable CO 2 Valves in an Ultrathin UiO-67 Membrane. Chem. Mater. 29, 3111–3117; 10.1021/acs.chemmater.7b00147 (2017).Tan, N. Y. et al. Investigation of the terahertz vibrational modes of ZIF-8 and ZIF-90 with terahertz time-domain spectroscopy. Chem Commun 51, 16037–16040; 10.1039/c5cc06455d (2015). Figure 1

Mapping molecular pathways for embryonic Sertoli cells derivation based on differentiation model of mouse embryonic stem cells

BackgroundEmbryonic Sertoli cells (eSCs) have been known for playing important roles in male reproductive development system. In current studies, eSCs were mainly generated from induced intermediate mesoderm. The deriving mechanism of eSCs has been unclear so far. Therefore, this work was aimed to reveal the molecular pathways during derivation of eSCs.MethodsIn this scenario, a differentiation model from mouse embryonic stem cells (mESCs) to eSCs was established through spatiotemporal control of 5 key factors, Wilms tumor 1 homolog (Wt1), GATA binding protein 4 (Gata4), nuclear receptor subfamily 5, group A, member 1 (Nr5a1, i.e., Sf1), SRY (sex determining region Y)-box 9 (Sox9), doublesex, and mab-3 related transcription factor 1 (Dmrt1). To investigate the molecular mechanism, these key factors were respectively manipulated through a light-switchable (light-on) system, tetracycline-switchable (Tet-on) system, and CRISPR/Cas9 knock out (KO) system.ResultsVia the established approach, some embryonic Sertoli-like cells (eSLCs) were induced from mESCs and formed ring-like or tubular-like structures. The key factors were respectively manipulated and revealed their roles in the derivation of these eSLCs. Based on these results, some molecular pathways were mapped during the development of coelomic epithelial somatic cells to eSCs.ConclusionsThis differentiation model provided a high controllability of some key factors and brought a novel insight into the deriving mechanism of Sertoli cells.

Dopant-induced photoresponsivity in coumarin-dye-sensitized nanowire NiO/p-Si heterojunction

This article presents dopant-induced photoresponsivity in Coumarin (CO)-dye-sensitized nanowire NiO/p-Si. The photoresponse modulation of Al/CO doped NiO/p-Si/Al heterojunction relates to different configurations of NiO under light. This performance is controlled by different concentration of CO-doping based on calcination and carbon (C) incorporation of NiO. Some electronic parameters are determined (the rectification ratio RR, the engineered ideality factor n and barrier height φb). CO doped NiO/p-Si photodiode can be designed as light-switchable systems with the tunable ON and OFF states. The capacitance (C)-time (s) measurements show that the heterojunction has photocapacitive behavior as a result of the doping amount of CO in NiO. Results show that we should pay more attention to the influence of CO dye on metal oxides and/or NiO for the optical switching devices and photodiode.

A Synthetic Light-switchable System based on CRISPR Cas13a Regulates the Expression of LncRNA MALAT1 and Affects the Malignant Phenotype of Bladder Cancer Cells.

DNA sequences drive their various functions through post-transcriptional processes, using mRNA or lncRNA (long non-coding RNA), and this accommodates the gene network by using various RNA types. However, the tools necessary to regulate RNA molecules are few. Likewise, RNA knockdown techniques that can be artificially controlled have not been extensively explored. Here, we investigated a novel light-inducible synthetic system based on CRISPR-Cas13a that can be used for RNA knockdown and binding in cancer cells. Based on the techniques of synthetic molecular biology, we constructed a light sensor, which efficiently induced Cas13a protein expression after blue light illumination. We also chose a lncRNA, Metastasis-associated Lung Adenocarcinoma Transcript 1 (MALAT1), as the functional target and detected it in bladder cancer 5637 and T24 cells in order to demonstrate the application of our synthetic system. Fluorescence reporter assays and real-time quantitative PCR (qRT-PCR) were used to detect the expression of the target gene. Phenotypic experiments were also used to test the effects of our synthetic system in bladder cancers. The results showed that our synthetic light-switchable system could inhibit the expression of MALAT1, and the fluorescence activity of enhanced green fluorescent protein. Our novel system provides a new technique to study RNA functions in gene networks and for precise tumor treatments.

Cell-Free Optogenetic Gene Expression System.

Optogenetic tools provide a new and efficient way to dynamically program gene expression with unmatched spatiotemporal precision. To date, their vast potential remains untapped in the field of cell-free synthetic biology, largely due to the lack of simple and efficient light-switchable systems. Here, to bridge the gap between cell-free systems and optogenetics, we studied our previously engineered one component-based blue light-inducible Escherichia coli promoter in a cell-free environment through experimental characterization and mathematical modeling. We achieved >10-fold dynamic expression and demonstrated rapid and reversible activation of the target gene to generate oscillatory response. The deterministic model developed was able to recapitulate the system behavior and helped to provide quantitative insights to optimize dynamic response. This in vitro optogenetic approach could be a powerful new high-throughput screening technology for rapid prototyping of complex biological networks in both space and time without the need for chemical induction.

Light-switchable systems for remotely controlled drug delivery

Light-switchable systems have recently received attention as a new mode of remotely controlled drug delivery. In the past, a multitude of nanomedicine studies have sought to enhance the specificity of drug delivery to target sites by focusing on receptors overexpressed on malignant cells or environmental features of diseases sites. Despite these immense efforts, however, there are few clinically available nanomedicines. We need a paradigm shift in drug delivery. One strategy that may overcome the limitations of pathophysiology-based drug delivery is the use of remotely controlled delivery technology. Unlike pathophysiology-based active drug targeting strategies, light-switchable systems are not affected by the heterogeneity of cells, tissue types, and/or microenvironments. Instead, they are triggered by remote light (i.e., near-infrared) stimuli, which are absorbed by photoresponsive molecules or three-dimensional nanostructures. The sequential conversion of light to heat or reactive oxygen species can activate drug release and allow it to be spatio-temporally controlled. Light-switchable systems have been used to activate endosomal drug escape, modulate the release of chemical and biological drugs, and alter nanoparticle structures to control the release rates of drugs. This review will address the limitations of pathophysiology-based drug delivery systems, the current status of light-based remote-switch systems, and future directions in the application of light-switchable systems for remotely controlled drug delivery.

A light-switchable bidirectional expression system in filamentous fungus Trichoderma reesei

The filamentous fungi Trichoderma reesei is widely used in the production of cellulolytic enzymes and recombinant proteins. However, only moderate success has been achieved in expressing heterologous proteins in T. reesei. Light-dependent control of DNA transcription, and protein expression have been demonstrated in bacteria, fungi, and mammalian cells. In this study, light inducible transactivators, a “light-on” bidirectional promoter and a “light-off” promoter were constructed successfully in T. reesei for the first time. Our light inducible transactivators can homodimerize and bind to the upstream region of artificial promoters to activate or repress genes transcription. Additionally, we upgraded the light-inducible system to on-off system that can simultaneously control the expression of multiple heterologous proteins in T. reesei. Moreover, a native cellulase-free background for the expression of heterologous proteins was achieved by knocking out the genes involved in transcriptional regulation and encoding of cellulases: xyr1, cbh1, and cbh2. Our light-switchable system showed a very little background protein expression and robust activation in the blue light with significantly improved heterologous protein expression. We demonstrate that our light-switchable system has a potential application as an on/off “switch” that can simultaneously regulate the expression of multiple genes in T. reesei under native cellulase-free background.

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

In recent years, it has been shown that LOV (light, oxygen, voltage) and BLUF (Blue Light sensing Using FAD) photosensory proteins are functioning as photoreceptors of light-regulated processes not only in eukaryotes but also in numerous prokaryotes. In bacterial photoreceptors, LOV and BLUF domains with attached flavin chromophores are often associated with different effector domains, which possess enzymatic and other functions, constituting modular light-switchable systems. At present, some progress in uncovering the photoactivation mechanisms of such systems has been achieved. They are based on the chromophore photoreaction-induced changes in the photosensory domain structures and subsequent signal transduction to the effector domains. Understanding of the signal transduction principles in LOV and BLUF photosensors is important for designing, on their basis, photo-switchable enzymes and transcriptional systems applied in optogenetics—a new field in cell biology and biotechnology. The structural aspects of signal transduction by light-activated LOV and BLUF photoreceptors and their regulatory functions in bacteria, as well as some recent advances in using LOV and BLUF photosensors as activators in optogenetic systems for regulation of cellular processes are discussed.

Bacterial photosensory proteins: Regulatory functions and optogenetic applications

Three classes of light-sensory regulatory proteins, which have been identified in genomes of numerous phototrophic and nonphotosynthetic bacteria, are discussed: the UVA/blue light sensitive BLUF and LOV domain-containing proteins and red/far-red light-sensitive phytochromes. Light perception by these chromoproteins is provided by the flavin or bilin (in phytochromes) chromophores binding to their photosensory domains. Bacterial photoreceptors also contain a variety of effector domains with enzymatic DNA-binding and other functions, which compose modular light-switchable systems. In recent years, progress was achieved in uncovering the photoactivation mechanisms of such systems. Based on the chromophore phototransformation-induced changes in the domain structures, these mechanisms cause the biochemical signal cascades which can control the light-dependent physiological responses of the cells. The new information obtained is important not only for understanding the fundamental mechanisms of light perception and signal transduction by bacterial photosensory proteins but also as a basis for designing photo-switchable enzymes and light-inducible gene expression systems, which may be used in optogenetics, a new field in cell biology and biotechnology. The presents review is focused on the structural aspects of signal transduction in light-activated bacterial photoreceptors, on their regulatory functions, and on some recent advances in using LOV and BLUF photosensors in optogenetics for the regulation of biological processes.

Switchable Gene Expression in Escherichia coli Using a Miniaturized Photobioreactor

We present a light-switchable gene expression system for both inducible and switchable control of gene expression at a single cell level in Escherichia coli using a previously constructed light-sensing system. The λ cI repressor gene with an LVA degradation tag was expressed under the control of the ompC promoter on the chromosome. The green fluorescent protein (GFP) gene fused to a λ repressor-repressible promoter was used as a reporter. This light-switchable system allows rapid and reversible induction or repression of expression of the target gene at any desired time. This system also ensures homogenous expression across the entire cell population. We also report the design of a miniaturized photobioreactor to be used in combination with the light-switchable gene expression system. The miniaturized photobioreactor helps to reduce unintended induction of the light receptor due to environmental disturbances and allows precise control over the duration of induction. This system would be a good tool for switchable, homogenous, strong, and highly regulatable expression of target genes over a wide range of induction times. Hence, it could be applied to study gene function, optimize metabolic pathways, and control biological systems both spatially and temporally.

Design of Beacon Light Switch Based on FPGA

A new design of using FPGA to realize array beacon light switching is preferred. Based on spatial diversity laser communication, the problem of multiplexing beacon light celerity and synchronism switching has been settled. Karnaugh map method was used in the paper to generate the time sequencing driving four motors, which was used in the schematic diagram design. Experiment results show that the system(or the design) in this paper cooperated with multiplexing beacon light has been realized, which simplifys the traditional beacon light switch system frame.