Scientific publications

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. © 2018, The Author(s).

Synthetic biology applies engineering concepts to build cellular systems that perceive and process information. This is achieved by assembling genetic modules according to engineering design principles. Recent advance in the field has contributed optogenetic switches for controlling diverse biological functions in response to light. Here, the concept is introduced to apply synthetic biology switches and design principles for the synthesis of multi-input-processing materials. This is exemplified by the synthesis of a materials system that counts light pulses. Guided by a quantitative mathematical model, functional synthetic biology-derived modules are combined into a polymer framework resulting in a biohybrid materials system that releases distinct output molecules specific to the number of input light pulses detected. Further demonstration of modular extension yields a light pulse-counting materials system to sequentially release different enzymes catalyzing a multistep biochemical reaction. The resulting smart materials systems can provide novel solutions as integrated sensors and actuators with broad perspectives in fundamental and applied research. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Molecular traps can control activity and abundance of many biological factors. Here, we report the development of a generic opto-trap to reversibly bind and release biomolecules with high spatiotemporal control by illumination with non-invasive and cell-compatible red and far-red light. We use the Arapidopsis thaliana photoreceptor phytochrome B to regulate the release of diverse proteins from a variety of material scaffolds. Fusion of a short 100 amino acids “PIF-tag” derived from the phytochrome interacting factor 6, renders arbitrary molecules opto-trap-compatible. Reversible opto-trapping of target molecules enables novel possibilities for future developments in diagnostics, therapeutics, and basic research. Statement of Significance: The investigation of cellular signaling events or the development of complex therapeutics and integrative diagnostic devices requires the deliberate control of biomolecule abundance and activity. During recent years, the use of natural photoreceptors within cells leveraged the control of diverse cellular events, benefiting from the superior spatial and temporal control characteristics of light as compared to conventional chemical stimuli. Concurrently, biological switches entailing intrinsic compatibility toward biological environments increasingly found application in biohybrid materials. We employ the plant red/far-red photoreceptor phytochrome B, which reversibly interacts with its phytochrome interacting factors (PIFs), for developing a generic opto-trap. This platform allows the use of red and far-red light to spatiotemporally control binding and release of arbitrary PIF-fused biomolecules from various material scaffolds. © 2018 Acta Materialia Inc.

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. Copyright © 2018 American Chemical Society.

Focal adhesion kinase (FAK) integrates signaling from integrins, growth factor receptors and mechanical stress to control cell adhesion, motility, survival and proliferation. Here, we developed a single-component, photo-activatable FAK, termed optoFAK, by using blue light-induced oligomerization of cryptochrome 2 (CRY2) to activate FAK-CRY2 fusion proteins. OptoFAK functions uncoupled from physiological stimuli and activates downstream signaling rapidly and reversibly upon blue light exposure. OptoFAK stimulates SRC creating a positive feedback loop on FAK activation, facilitating phosphorylation of paxillin and p130Cas in adherent cells. In detached cells or in mechanically stressed adherent cells, optoFAK is autophosphorylated upon exposure to blue light, however, downstream signaling is hampered indicating that the accessibility to these substrates is disturbed. OptoFAK may prove to be a useful tool to study the biological function of FAK in growth factor and integrin signaling, tension-mediated focal adhesion maturation or anoikis and could additionally serve as test system for kinase inhibitors. © 2017

OptoBase is an online platform for molecular optogenetics. At its core is a hand-annotated and ontology-supported database that aims to cover all existing optogenetic switches and publications, which is further complemented with a collection of convenient optogenetics-related web tools. OptoBase is meant both for expert optogeneticists to easily keep track of the field, as well as for all researchers who find optogenetics inviting as a powerful tool to address their biological questions of interest. It is available at https://www.optobase.org. This work also presents OptoBase-based analysis of the trends in molecular optogenetics. © 2018 American Chemical Society.

In plants, the phytohormone auxin acts as a master regulator of developmental processes and environmental responses. The best characterized process in the auxin regulatory network occurs at the subcellular scale, wherein auxin mediates signal transduction into transcriptional programs by triggering the degradation of Aux/IAA transcriptional repressor proteins in the nucleus. However, whether and how auxin movement between the nucleus and the surrounding compartments is regulated remain elusive. Using a fluorescent auxin analog, we show that its diffusion into the nucleus is restricted. By combining mathematical modeling with time course assays on auxin-mediated nuclear signaling and quantitative phenotyping in single plant cell systems, we show that ER-to-nucleus auxin flux represents a major subcellular pathway to directly control nuclear auxin levels. Our findings propose that the homeostatically regulated auxin pool in the ER and ER-to-nucleus auxin fluxes underpin auxin-mediated downstream responses in plant cells. Middleton et al. study how the plant phytohormone auxin enters the nucleus by using quantitative phenotyping in single plant cell systems and bespoke mathematical models that relate controlled perturbations to experimentally measurable responses. Their findings show that auxin predominantly enters the nucleus via the endoplasmic reticulum. © 2018 The Authors

We here present a method that combines genetic code expansion with CRISPR/Cas9 genome engineering to label endogenously expressed proteins with high spatiotemporal resolution. The method exploits the use of an orthogonal tRNA/tRNA synthetase pair in conjugation with noncanonical amino acids to create stop codon read through events. To demonstrate the functionality of the method, we pulse labeled endogenous β-actin and tumor protein p53 with a minimally invasive HA tag at their C-termini. Targeting the protein label with a proximity ligation assay plus real time imaging facilitates seamless quantification of the protein synthesis rate and spatial localization at the single cell level. The presented approach does not interfere with any physiological control of cellular expression, nor did we observe any perturbation of endogenous protein functions. © 2018 American Chemical Society.

The translation of engineering designs to materials sciences by means of synthetic biological tools represents a novel concept for the development of information-processing materials systems. Here, we provide data on the mathematical model-guided implementation of a biomaterials-based positive feedback loop for the detection of proteolytic activities. Furthermore, we present data on an extended system design for the detection of the antibiotic novobiocin. This work is related to the research article “Synthetic biology-inspired design of signal-amplifying materials systems” (Wagner et al., 2018) [1]. © 2018 The Authors

Nanobodies, the smallest possible antibody format, have become of considerable interest for biotechnological and immunotherapeutic applications. They show excellent robustness, are non-immunogenic in humans, and can easily be engineered and produced in prokaryotic hosts. Traditionally, nanobodies are selected from camelid immune libraries involving the maintenance and treatment of animals. Recent advances have involved the generation of nanobodies from naïve or synthetic libraries. However, such approaches demand large library sizes and sophisticated selection procedures. Here, we propose an alternative, two-step approach for the design and generation of nanobodies. In a first step, complementarity-determining regions (CDRs) are grafted from conventional antibody formats onto nanobody frameworks, generating weak antigen binders. In a second step, the weak binders serve as templates to design focused synthetic phage libraries for affinity maturation. We validated this approach by grafting toxin-and hapten-specific CDRs onto frameworks derived from variable domains of camelid heavy-chain-only antibodies (VHH). We then affinity matured the hapten binder via panning of a synthetic phage library. We suggest that this strategy can complement existing immune, naïve, and synthetic library based methods, requiring neither animal experiments, nor large libraries, nor sophisticated selection protocols. © 2018 by the authors. Licensee MDPI, Basel, Switzerland.