Materialorientierte Synthetische Biologie

Optogenetic approaches have gathered momentum in precisely modulating and interrogating cellular signalling and gene expression. The use of optogenetics on the outer cell surface to interrogate how cells receive stimuli from their environment, however, has so far not reached its full potential. Here we demonstrate the development of an optogenetically regulated membrane receptor-ligand pair exemplified by the optically responsive interaction of an integrin receptor with the extracellular matrix. The system is based on an integrin engineered with a phytochrome-interacting factor domain (OptoIntegrin) and a red light-switchable phytochrome B-functionalized matrix (OptoMatrix). This optogenetic receptor-ligand pair enables light-inducible and -reversible cell-matrix interaction, as well as the controlled activation of downstream mechanosensory signalling pathways. Pioneering the application of optogenetic switches in the extracellular environment of cells, this OptoMatrix–OptoIntegrin system may serve as a blueprint for rendering matrix–receptor interactions amendable to precise control with light. © 2019, The Author(s).

Noncoding small RNAs, such as microRNAs, are becoming the biomarkers of choice for multiple diseases in clinical diagnostics. A dysregulation of these microRNAs can be associated with many different diseases, such as cancer, dementia, and cardiovascular conditions. The key for effective treatment is an accurate initial diagnosis at an early stage, improving the patient's survival chances. In this work, the first clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a-powered microfluidic, integrated electrochemical biosensor for the on-site detection of microRNAs is introduced. Through this unique combination, the quantification of the potential tumor markers microRNA miR-19b and miR-20a is realized without any nucleic acid amplification. With a readout time of 9 min and an overall process time of less than 4 h, a limit of detection of 10 pm is achieved, using a measuring volume of less than 0.6 µL. Furthermore, the feasibility of the biosensor platform to detect miR-19b in serum samples of children, suffering from brain cancer, is demonstrated. The validation of the obtained results with a standard quantitative real-time polymerase chain reaction method shows the ability of the electrochemical CRISPR-powered system to be a low-cost, easily scalable, and target amplification-free tool for nucleic acid based diagnostics. © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Multiprotein complexes control the behavior of cells, such as of lymphocytes of the immune system. Methods to affinity purify protein complexes and to determine their interactome by mass spectrometry are thus widely used. One drawback of these methods is the presence of false positives. In fact, the elution of the protein of interest (POI) is achieved by changing the biochemical properties of the buffer, so that unspecifically bound proteins (the false positives) may also elute. Here, we developed an optogenetics-derived and light-controlled affinity purification method based on the light-regulated reversible protein interaction between phytochrome B (PhyB) and its phytochrome interacting factor 6 (PIF6). We engineered a truncated variant of PIF6 comprising only 22 amino acids that can be genetically fused to the POI as an affinity tag. Thereby the POI can be purified with PhyB-functionalized resin material using 660 nm light for binding and washing, and 740 nm light for elution. Far-red light-induced elution is effective but very mild as the same buffer is used for the wash and elution. As proof-of-concept, we expressed PIF-tagged variants of the tyrosine kinase ZAP70 in ZAP70-deficient Jurkat T cells, purified ZAP70 and associating proteins using our light-controlled system, and identified the interaction partners by quantitative mass spectrometry. Using unstimulated T cells, we were able to detect the known interaction partners, and could filter out all other proteins. Copyright © 2019 Hörner, Eble, Yousefi, Schwarz, Warscheid, Weber and Schamel. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Phytochromes are important photoreceptors of plants, bacteria, and fungi responsive to light in the red and far-red spectrum. For increasing applications in basic research, synthetic biology, and materials sciences, it is required to recombinantly produce and purify phytochromes in high amounts. An ideal host organism for this purpose is E. coli due to its widespread use, fast growth, and ability for high-cell-density fermentation. Here, we describe the development of a generic platform for the production of phytochromes in E. coli that is compatible with high-cell-density fermentation. We exemplify our approach by the production of the photosensory domains of phytochrome B (PhyB) from A. thaliana and of the cyanobacterial phytochrome 1 (Cph1) from Synechocystis PCC 6803 in the multigram scale per 10 L fermentation run. Copyright © 2019 American Chemical Society.

As the field of tissue engineering develops, methods for screening combinations of signals for their effects on stem cell behavior are needed. We introduce a microgel-based screening platform for testing combinations of in situ-generated proteins on stem cell fate in ultrahigh-throughput. Compartmentalizing individual sets of growth factors was addressed by encapsulating aggregates of stable recombinant cell lines secreting individual glycoproteins into microgels through an on-chip polymerization. When these 'microniches' are cultured with a cell type of interest, fluorescence reporters indicate positive niches that perform the desired function, and the underlying producer cell lines of these selected microniches are analyzed by barcoded RNA sequencing. The microniche-based screening work-flow was validated via a model system based on engineered mammalian cells expressing yellow fluorescent protein (YFP) upon anti-inflammatory cytokine interleukin 4 (IL4)-based activation. © 2019 The Royal Society of Chemistry.

Interrogation and control of cellular fate and function using optogenetics is providing revolutionary insights into biology. Optogenetic control of cells is achieved by coupling genetically encoded photoreceptors to cellular effectors and enables unprecedented spatiotemporal control of signaling processes. Here, a fast and reversibly switchable photoreceptor is used to tune the mechanical properties of polymer materials in a fully reversible, wavelength-specific, and dose- and space-controlled manner. By integrating engineered cyanobacterial phytochrome 1 into a poly(ethylene glycol) matrix, hydrogel materials responsive to light in the cell-compatible red/far-red spectrum are synthesized. These materials are applied to study in human mesenchymal stem cells how different mechanosignaling pathways respond to changing mechanical environments and to control the migration of primary immune cells in 3D. This optogenetics-inspired matrix allows fundamental questions of how cells react to dynamic mechanical environments to be addressed. Further, remote control of such matrices can create new opportunities for tissue engineering or provide a basis for optically stimulated drug depots. © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Subcellular localization of signal molecules is a hallmark in organizing the signaling network. OpEn-Tag is a modular optogenetic endomembrane targeting toolbox that allows alteration of the localization and therefore the activity of signaling processes with the spatiotemporal resolution of optogenetics. OpEn-Tag is a two-component system employing (1) a variety of targeting peptides fused to and thereby dictating the localization of mCherry-labeled cryptochrome 2 binding protein CIBN toward distinct endomembranes and (2) the cytosolic, fluorescence-labeled blue light photoreceptor cryptochrome 2 as a customizable building block that can be fused to proteins of interest. The combination of OpEn-Tag with growth factor stimulation or the use of two membrane anchor sequences allows investigation of multilayered signal transduction processes as demonstrated here for the protein kinase AKT. © 2019 American Chemical Society.

The exploitation of nanosized materials for the delivery of therapeutic agents is already a clinical reality and still holds unrealized potential for the treatment of a variety of diseases. This review discusses physiological barriers a nanocarrier must overcome in order to reach its target, with an emphasis on cancer nanomedicine. Stages of delivery include residence in the blood stream, passive accumulation by virtue of the enhanced permeability and retention effect, diffusion within the tumor lesion, cellular uptake, and arrival at the site of action. We also briefly outline strategies for engineering nanoparticles to more efficiently overcome these challenges: Increasing circulation half-life by shielding with hydrophilic polymers, such as PEG, the limitations of PEG and potential alternatives, targeting and controlled activation approaches. Future developments in these areas will allow us to harness the full potential of nanomedicine. © Copyright © 2019 Thomas and Weber.

Synthetic biology applies engineering concepts to build cells that perceive and process information. Examples include cells engineered to perform basic digital or analog computation. These circuits serve as basis for the construction of complex integrated cellular networks that offer manifold applications in fundamental and applied research. Here, we introduce the concept of using design approaches and molecular tools applied in synthetic biology for the construction of interconnected biohybrid materials systems with information processing functionality. We validate this concept by modularly assembling protein and polymer building blocks to generate stimulus-responsive materials. Guided by a quantitative mathematical model, we next interconnect these materials into a materials system that acts as both a signal detector and as an amplifier based on a built-in positive feedback loop. The functionality and versatility of this materials system is demonstrated by the detection of enzymatic activities and drugs. The modular design concept presented here thus represents a blueprint for integrating synthetic biology-inspired information-processing circuits into polymer materials. As integrated sensors and actuators, the resulting smart materials systems could provide novel solutions with broad perspectives in research and development. © 2018 Elsevier Ltd

Feedforward and feedback loops are key regulatory elements in cellular signaling and information processing. Synthetic biology exploits these elements for the design of molecular circuits that enable the reprogramming and control of specific cellular functions. These circuits serve as a basis for the engineering of complex cellular networks, opening the door for numerous medical and biotechnological applications. Here, a similar principle is applied. Feedforward and positive feedback circuits are incorporated into biohybrid polymer materials in order to develop signal-sensing and signal-processing devices. This concept is exemplified by the detection of the proteolytic activity of the botulinum neurotoxin A. To this aim, site-specific proteases are incorporated into receiver, transmitter, and output materials, and their release, diffusion, and/or activation are wired according to a feedforward or a positive feedback circuit. The development of a quantitative mathematical model enables analysis and comparison of the performance of both systems. The flexible design could be easily adapted to detect other toxins or molecules of interest. Furthermore, cellular signaling or gene regulatory pathways could provide additional blueprints for the development of novel biohybrid circuits. Such information-processing, material-embedded biological circuits hold great promise for a variety of analytical, medical, or biotechnological applications. © 2018 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim