Materialorientierte Synthetische Biologie

The recent implementation of various high-throughput biochemical and bioanalytical platforms for the study of biological systems has resulted in a wealth of experimental information that systems biology integrates into models and functional descriptions of organisms. The fast tempo of systems biology development is currently bringing in a revolution in the understanding of cell networks by providing with a holistic comprehension of cellular components and their interaction dynamics. This thorough description of biological systems has laid the grounds for the development of synthetic biology, a discipline applying basic principles of engineering for the rational assembly of biological modules into higher order complex biological systems with desired properties. Despite the success of this new field for the generation of biotechnological tools, it has not been yet widely applied to plant systems. This review aims at describing the current status of systems biology, its contribution to our understanding of plant metabolism, expression and regulatory networks and how synthetic biology approaches could benefit utilising plant and bacterial 'omics' as a source for the design and development of biological modules for the improvement of plant stress tolerance and crop production, among other applications. The article further describes synthetic biology strategies currently being applied to plant metabolic engineering, development of signalling pathways and synthetic organelles, and the potential of this new field for the understanding of plant cellular functioning and the generation of plant biotechnological tools. © 2012 Elsevier B.V..

Interactive materials that specifically respond to environmental stimuli hold high promise as energy-autonomous sensors and actuators in biomedicine, analytics or microsystems engineering. However, the implementation of materials specifically responsive to a given small molecule has so far been hampered by a lack of generically applicable stimulus sensors. In this study, a novel and likely general strategy for the synthesis of biohybrid materials with desired stimulus specificity is established. The strategy is based on allosterically regulated DNA-binding proteins, a conserved protein family that has evolved in prokaryotes to sense and respond to most diverse molecules in order to enable bacterial survival in a changing environment. The novel hydrogel design concept is demonstrated with the example of single-chain TetR, a protein that binds the tetO DNA motif and dissociates thereof in the presence of the antibiotic tetracycline. Therefore, linear polyacrylamide is crosslinked via the TetR/tetO interaction to a biohybrid material that can subsequently be dissolved by tetracycline in a dose-dependent manner. This drug-induced dissolution is applied for the adjustable release of the cytokine interleukin 4 in a tetracycline-dependent manner. The design concept developed in this study might serve as a blueprint for the synthesis of biohybrid materials responsive to drugs, metabolites or toxins by replacing TetR/tetO with another protein/DNA pair showing the desired stimulus specificity. A biohybrid hydrogel is synthesized for the drug-inducible release of biopharmaceuticals. The hydrogel consists of linear polyacrylamide crosslinked by the interaction of the tetracycline repressor scTetR with its target DNA sequence tetO. Addition of tetracycline dissociates the protein-DNA interaction and triggers the release of a previously embedded payload protein. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inducible expression systems represent the founding technology for the emergence of synthetic biology in mammalian cells. The core molecules in these systems are bacterial regulator proteins that bind to or dissociate from a cognate DNA operator sequence in response to an exogenous stimulus like a small-molecule inducer. In this chapter, we describe a generic protocol of how bacterial regulator proteins can be applied to the design, construction, and optimization of an inducible expression system in mammalian cells. By choosing regulator proteins with an appropriate small-molecule inducer, this protocol provides a straightforward approach for establishing biosensors, cell-to-cell communication systems, or tools to control gene expression in vivo. © 2011 Elsevier Inc. All rights reserved.

The rapid development of synthetic biology is a paradigm of how the molecular diversity of naturally occurring gene control components can be used to design synthetic control devices and gene networks that provide precisely programmed transgene expression dynamics in space and time. Here we offer an overview on recent advances in the modular design of trigger-inducible mammalian expression devices that are either responsive by exogenous stimuli such as chemicals and physical cues or controlled by endogenous metabolites driving prosthetic circuits to treat metabolic disorders in a self-sufficient manner. Compatible genetic switches can also be assembled to synthetic gene networks that show highly complex expression dynamics such as temporally resolved band-detect functions or oscillating transgene expression profiles. The ongoing metagenomic discovery and characterization of the unexplored sequence space is constantly increasing the molecular diversity in fundamental control components that fuels the further development of synthetic biology. © 2011 Elsevier Ltd.

Synthetic quorum-sensing systems in mammalian cells has enabled the implementation of time- and distance-dependent bioprocesses, as well as the design of synthetic ecosystems emulating clinically important host–parasite interactions. In this chapter, we provide a detailed protocol of the design of a mammalian cell-to-cell signaling device and its integration into a mammalian quorum-sensing system for cell density-induced expression product genes. Cell-to-cell signaling is based on a sender cell, metabolically engineered for expression of alcohol dehydrogenase converting ethanol into acetaldehyde, and a receiver cell line for the dose-dependent translation of the acetaldehyde concentration into transgene expression by an acetaldehyde-responsive promoter. This protocol can be adapted easily to various cell types and transgenes for the design of versatile mammalian cell-based quorum-sensing systems. © 2011, Springer Science+Business Media, LLC.

Synthetic Biology aims at the design and construction of biologic systems with desired features by applying a modular strategy. This approach was used to investigate the interaction of multiple organisms in synthetic ecosystems.

Gene therapy scientists have developed expression systems for therapeutic transgenes within patients, which must be seamlessly integrated into the patient's physiology by developing sophisticated control mechanisms to titrate expression levels of the transgenes into the therapeutic window. However, despite these efforts, gene-based medicine still faces security concerns related to the administration of the therapeutic transgene vector. Here, molecular tools developed for therapeutic transgene expression can readily be transferred to materials science to design a humanized drug depot that can be implanted into mice and enables the trigger-inducible release of a therapeutic protein in response to a small-molecule inducer. The drug depot is constructed by embedding the vascular endothelial growth factor (VEGF121) as model therapeutic protein into a hydrogel consisting of linear Polyacrylamide crosslinked with a homodimeric variant of the human FKbinding protein 12 (F M), originally developed for gene therapeutic applications, as well as with dimethylsuberimidate. Administrating increasing concentrations of the inducer molecule FK506 triggers the dissociation of FM thereby loosening the hydrogel structure and releasing the VEGF121 payload in a dose-adjustable manner. Subcutaneous implantation of the drug depot into mice and subsequent administration of the inducer by injection or by oral intake triggers the release of VEGF121 as monitored in the mouse serum. This study is the first demonstration of a stimuli-responsive hydrogel that can be used in mammals to release a therapeutic protein on demand by the application of a small-molecule stimulus. This trigger-inducible release is a starting point for the further development of externally controlled drug depots for patient-compliant administration of biopharmaceuticals. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthetic biology as the discipline of reconstructing natural and designing novel biological systems is gaining increasing impact in signaling science. This review article provides insight into synthetic approaches for analyzing and synthesizing signaling processes starting with strategies into how natural and pathological signaling pathways can be reconstructed in an evolutionary distant host to study their topology and function while avoiding interference with the original host background. In the second part we integrate synthetic strategies in the rewiring of signaling systems at the nucleic acid and protein level to reprogram cellular functions for biotechnological applications. The last part focuses on synthetic inter-cell and inter-species signaling devices and their integration into synthetic ecosystems to study fundamental mechanisms governing the co-existence of species. We finally address current bottlenecks in the (re-)design of signaling pathways and discuss future directions in signaling-related synthetic biology. © 2010 The Royal Society of Chemistry.

The design and construction of synthetic gene circuits with complex spatiotemporal dynamics was pioneered in bacteria, but it took almost a decade until synthetic biologists were able to construct genetic circuits with complex spatiotemporal dynamics in mammalian cells. This review highlights the most recent advances in mammalian synthetic biology, and it describes metabolite, hormone, and light-triggered genetic switches as well as the design and construction of synthetic networks that feature tunable oscillations. We conclude by discussing not only the current limitations but also possible ways to transform the construction of synthetic mammalian systems from an art into a predictive engineering discipline. © 2010 Elsevier Ltd.

Inducer-dependent prokaryotic transcriptional repressor proteins that originally evolved to orchestrate the transcriptome with intracellular and extracellular metabolite pools, have become universal tools in synthetic biology, drug discovery, diagnostics and functional genomics. Production of the repressor proteins is often limited due to inhibiting effects on the production host and requires iterative process optimization for each individual repressor. At the example of the Streptomyces pristinaespiralis-derived streptogramin-dependent repressor PIP, the expression of which was shown to inhibit growth of Escherichia coli BL21*, we demonstrate that the addition of the PIP-specific streptogramin antibiotic pristinamycin I neutralizes the growth-inhibiting effect and results in >100-fold increased PIP titers. The yield of PIP was further increased 2.5-fold by the engineering of a new E. coli host suitable for the production of growth-inhibiting proteins encoded by an unfavorable codon usage. PIP produced in the presence of pristinamycin I was purified and was shown to retain the antibiotic-dependent binding to its operator pir as demonstrated by a fluorescence resonance energy transfer (FRET)-based approach. At the example of the macrolide-, tetracycline- and arsenic-dependent repressors MphR(A), TetR and ArsR, we further demonstrate that the production yields can be increased 2- to 3-fold by the addition of the cognate inducer molecules erythromycin, tetracycline and As3+, respectively. Therefore, the addition of inducer molecules specific to the target repressor protein seems to be a general strategy to increase the yield of this interesting protein class. © 2009 Elsevier Inc. All rights reserved.