Materialorientierte Synthetische Biologie

The ability to control mammalian genes in a synergistic mode using synthetic transcription factors is highly desirable in fields of tissue engineering, stem cell reprogramming and fundamental research. In this study, we developed a standardized toolkit utilizing an engineered CRISPR/Cas9 system that enables customizable gene regulation in mammalian cells. The RNA-guided dCas9 protein was implemented as a programmable transcriptional activator or repressor device, including targeting of endogenous loci. For facile assembly of single or multiple CRISPR RNAs, our toolkit comprises a modular RNAimer plasmid, which encodes the required noncoding RNA components. © 2014 American Chemical Society.

Chemically triggered molecular switches for controlling the fate and function of biological systems are fundamental to the emergence of synthetic biology and the development of biomedical applications. We here present the first chemically triggered switch for controlling the infectivity of adeno-associated viral (AAV) vectors. © 2014 The Royal Society of Chemistry.

Biohybrid hydrogels that change their mechanical properties in response to pharmacological cues hold high promises as externally controlled drug depots for biomedical applications. In this study, we devise a generically applicable method for the synthesis of micrometer-scale, injection-ready biohybrid materials. We use droplet-based microfluidics to generate monodisperse pre-microgel fluid droplets, wherein which we react fluorescein-modified 8-arm poly(ethylene glycol) with a thiol-functionalized humanized anti-fluorescein single chain antibody fragment and vinylsulfonefunctionalized 8-arm poly(ethylene glycol), resulting in the formation of stable, narrowly dispersed supramolecular microgels (30 and 150μm diameter). We demonstrate that the addition of free fluorescein to these microgels results in a weakening of their hydrogel structure, eventually leading to its disintegration. This method of formation of pharmacologically responsive biohybrid hydrogels in an injection-ready formulation is a pioneering example of a general approach for the synthesis of biohybrid hydrogel-based drug depots for biomedical applications. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Steadily growing demands for identification and quantification of cellular metabolites in higher throughput have brought a need for new analytical technologies. Here, we developed a synthetic biological sensor system for quantifying metabolites from biological cell samples. For this, bacterial transcription factors were exploited, which bind to or dissociate from regulatory DNA elements in response to physiological changes in the cellular metabolite concentration range. Representatively, the bacterial pyruvate dehydrogenase (PdhR), trehalose (TreR), and l-arginine (ArgR) repressor proteins were functionalized to detect pyruvate, trehalose-6-phosphate (T6P), and arginine concentration in solution. For each transcription factor the mutual binding behavior between metabolite and DNA, their working range, and othogonality were determined. High-throughput, parallel processing, and automation were achieved through integration of the metabolic sensor system on a microfluidic large-scale integration (mLSI) chip platform. To demonstrate the functionality of the integrated metabolic sensor system, we measured diurnal concentration changes of pyruvate and the plant signaling molecule T6P within cell etxracts of Arabidopsis thaliana rosettes. The transcription factor sensor system is of generic nature and extendable on the microfluidic chip. (Figure Presented). © 2014 American Chemical Society.

On command control of gene expression in time and space is required for the comprehensive analysis of key plant cellular processes. Even though some chemical inducible systems showing satisfactory induction features have been developed, they are inherently limited in terms of spatiotemporal resolution and may be associated with toxic effects. We describe here the first synthetic light-inducible system for the targeted control of gene expression in plants. For this purpose, we applied an interdisciplinary synthetic biology approach comprising mammalian and plant cell systems to customize and optimize a split transcription factor based on the plant photoreceptor phytochrome B and one of its interacting factors (PIF6). Implementation of the system in transient assays in tobacco protoplasts resulted in strong (95-fold) induction in red light (660 nm) and could be instantaneously returned to the OFF state by subsequent illumination with far-red light (740 nm). Capitalizing on this toggle switch-like characteristic, we demonstrate that the system can be kept in the OFF state in the presence of 740 nm-supplemented white light, opening up perspectives for future application of the system in whole plants. Finally we demonstrate the system's applicability in basic research, by the light-controlled tuning of auxin signalling networks in N. tabacum protoplasts, as well as its biotechnological potential for the chemical-inducer free production of therapeutic proteins in the moss P. patens. This journal is © the Partner Organisations 2014.

Light-triggered gene expression systems offer an unprecedented spatiotemporal resolution that cannot be achieved with classical chemically inducible genetic tools. Here we describe a protocol for red light-responsive gene expression in mammalian cells. This system can be toggled between stable ON and OFF states by short pulses of red and far-red light, respectively. In the protocol, CHO-K1 cells are transfected to allow red light-inducible expression of the secreted alkaline phosphatase (SEAP) reporter, and gene expression is tuned by illumination with light of increasing wavelengths. As a starting point for elaborate red light-responsive gene expression, we outline the reversible activation of gene expression and describe how a spatial pattern can be 'printed' on a monolayer of cells by using a photomask. The core protocol requires only 4 d from seeding of the cells to reporter quantification, and other than light-emitting diode (LED) illumination boxes no elaborate equipment is required. © 2014 Nature America, Inc.

Mechanobiology is a scientific interface discipline emerging from engineering and biology. With regard to tissue-regenerative cell-based strategies, mechanobiological concepts, including biomechanics as a target for cell and human mesenchymal stem cell behaviour, are on the march. Based on the periodontium as a paradigm, this mini-review discusses the key role of focal-adhesion kinase (FAK) in mechanobiology, since it is involved in mediating the transformation of environmental biomechanical signals into cell behavioural responses via mechanotransducing signalling cascades. These processes enable cells to adjust quickly to environmental cues, whereas adjustment itself relies on the specific intramolecular phosphorylation of FAK tyrosine residues and the multiple interactions of FAK with distinct partners. Furthermore, interaction-triggered mechanotransducing pathways govern the dynamics of focal adhesion sites and cell behaviour. Facets of behaviour not only include cell spreading and motility, but also proliferation, differentiation and apoptosis. In translational terms, identified and characterized biomechanical parameters can be incorporated into innovative concepts of cell- and tissue-tailored clinically applied biomaterials controlling cell behaviour as desired.

Light-dependent dimerization is the basis for recently developed noninvasive optogenetic tools. Here we present a novel tool combining optogenetics with the control of protein kinase activity to investigate signal transduction pathways. Mediated by Arabidopsis thaliana photoreceptor cryptochrome 2, we activated the protein kinase C-RAF by blue light-dependent dimerization, allowing for decoupling from upstream signaling events induced by surface receptors. The activation by light is fast, reversible, and not only time but also dose dependent as monitored by phosphorylation of ERK1/2. Additionally, light-activated C-RAF controls serum response factor-mediated gene expression. Light-induced heterodimerization of C-RAF with a kinase-dead mutant of B-RAF demonstrates the enhancing role of B-RAF as a scaffold for C-RAF activity, which leads to the paradoxical activation of C-RAF found in human cancers. This optogenetic tool enables reversible control of protein kinase activity in signal duration and strength. These properties can help to shed light onto downstream signaling processes of protein kinases in living cells. © 2013 American Chemical Society.

Prokaryotic transcriptional regulatory elements are widely utilized building blocks for constructing regulatory genetic circuits adapted for mammalian cells and have found their way into a broad range of biotechnological applications. Prokaryotic transcriptional repressors, fused to eukaryotic transactivation or repression domains, compose the transcription factor, which binds and adjusts transcription from chimeric promoters containing the repressor-specific operator sequence. Escherichia coli and Chlamydia trachomatis share common features in the regulatory mechanism of the biosynthesis of l-tryptophan. The repressor protein TrpR of C. trachomatis regulates the trpRBA operon and the TrpR of E. coli regulates the trpEDCBA operon, both requiring l-tryptophan as a co-repressor. Fusion of these bacterial repressors to the VP16 transactivation domain of Herpes simplex virus creates synthetic transactivators that could bind and activate chimeric promoters, assembled by placing repressor-specific operator modules adjacent to a minimal promoter, in an l-tryptophan-adjustable manner. Combinations of different transactivator and promoter variants from the same or different bacterial species resulted in a multitude of regulatory systems where l-tryptophan regulation properties, background noise, and maximal gene expression levels were significantly diverse. Different l-tryptophan analogues showed diverse regulatory capacity depending on the promoter/transactivator combination. We believe the systems approach to rationally choose promoters, transactivators and inducer molecules, to obtain desired and predefined genetic expression dynamics and control profiles, will significantly advance the design of new regulatory circuits as well as improving already existing ones. © 2012 Elsevier Inc.

Functional biomaterials that detect and correct pathological parameters hold high promises for biomedical application. In this study we describe a biohybrid hydrogel that detects elevated concentrations of uric acid and responds by dissolution and the release of uric acid-degrading urate oxidase. This material was synthesized by incorporating PEG-stabilized urate oxidase into a polyacrylamide hydrogel that was crosslinked by the uric acid-sensitive interaction between the uric acid transcription factor HucR and its operator hucO. We characterize the uric acid responsiveness of the material and demonstrate that it can effectively be applied to counteract flares of uric acid in a mouse model. This approach might be a first step towards a biomedical device autonomously managing uric acid burst associated to gouty arthritis and the tumor lysis syndrome. © 2013 Elsevier B.V. All rights reserved.