Materialorientierte Synthetische Biologie

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.

Hydrogels provide a highly favorable matrix for immobilizing growth factors, enzymes or cells for biomedical applications like tissue engineering, drug delivery or the treatment of metabolic diseases. In this study we describe the synthesis and characterization of a hydrogel able to degrade l-ornithine, a metabolite that is highly elevated in congenital hyperornithinemia. The hydrogel was synthesized by embedding the l-ornithine-degrading enzymes l-ornithine aminotransferase (OAT) and l-ornithine decarboxylase (ODC) into a polymer network. The network was formed from linear polyacrylamide crosslinked by heterodimers of ODC and ornithine decarboxylase antizyme (OAz). The resulting hydrogel was shown to be stable under physiological conditions and to efficiently degrade l-ornithine. The hydrogel-stabilizing ODC-OAz interactions could subsequently be dissociated by the addition of antizyme inhibitor (AzI) which resulted in the inducible dissolution of the hydrogel. This l-ornithine-degrading hydrogel that can efficiently be eliminated when its functionality is no longer required might represent a first step towards an enzyme substitution approach against hyperornithinemia. © 2012 Elsevier B.V.

The synthetic reconstruction of natural gene networks and the de novo design of artificial genetic circuits provide new insights into the cell's regulatory mechanisms and will open new opportunities for drug discovery and intelligent therapeutic schemes. We will present how modular synthetic biology tools like repressors, promoters and enzymes can be assembled into complex systems in order to discover small molecules to shut off antibiotic resistance in tubercle bacteria and to design self-sufficient therapeutic networks. The transfer of these synthetic biological modules to the materials science field enables the construction of novel drug-inducible biohybrid materials for biomedical applications. © 2012 Elsevier Inc.

Remote-controlled drug depots represent a highly valuable tool for the timely controlled administration of pharmaceuticals in a patient compliant manner. Here, the first pharmacologically controlled material that allows for the scheduled induction of a medical response in mice is described. To this aim, a novel, humanized biohybrid material that releases its cargo in response to a small-molecule stimulus licensed for human use is developed. The functionality of the material in mice is demonstrated by the remote-controlled delivery of a vaccine against the oncogenic human papillomavirus type 16. It is shown that the biohybrid depot-mediated immunoprotection is equivalent to the classical multi-injection-based vaccination. These results indicate that this material can be used as a universal remote-controlled vehicle for the patient-compliant delivery of vaccines and pharmaceuticals. A pharmacologically controlled hydrogel depot is presented allowing for the scheduled induction of a medical response in vivo. The vaccine-loaded hydrogel depot is administered to mice. At the desired point in time, the vaccine can be released from the depot by the oral administration of the stimulus molecule fluorescein resulting in protective immunization. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biohybrid materials combining synthetic polymers with biological components are highly suited for tissue engineering in order to emulate the behavior of natural materials such as the extracellular matrix (ECM). In order to allow for an optimal cell-material interplay, the physical and biological parameters of the artificial matrix need to be dynamically remodeled during cultivation. Current tissue engineering concepts are mainly based on passive remodeling mechanisms including the degradation of the hydrogel and the release of incorporated biomolecules and therefore do not enable external adjustment of cultivation conditions. We present a novel hydrogel material that is able to serve as a cell growth matrix, whose degradation and presentation of cell-interacting biomolecules can be externally controlled by the addition of a pharmacological substance. The hydrogel is based on branched polyethylene glycol that is covalently decorated with the aminocoumarin-antibiotic switchable gyrase B protein conferring stimulus-responsive degradation. ECM properties were conferred to the hydrogels with cell attachment motifs and a general approach for the incorporation and inducible release of therapeutic biomolecules. This smart biohybrid material has the potential to serve as a next-generation tissue engineering device which allows for dynamic external adjustment of the physical and biological parameters, resulting in optimally controlled tissue formation. © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.