Publikationen

The precise manipulation of growth factor signaling is central to the progress of tissue engineering. Methods for direct time-resolved activation of signaling pathways through controlled receptor dimerization have been reported; however, these suffer from the risks associated with gene transfer. Here we present an alternative gene transfer-free approach in the form of a protein switch featuring pharmacologically controlled ON-OFF regulation of growth factor activity. The reversible operation of the switch enables stimulation of target processes within a defined period of time. The protein switch provides a means for both studying and manipulating signaling processes, and is thus believed to be a valuable tool for basic research as well as tissue engineering and biomedical applications.

Detecting drug-target interactions in real-time is a powerful approach for drug discovery and analytics. We show here for the first time the ultra fast electrical real-time detection and quantification of antibiotics using a novel biohybrid nanosensor. The biomolecular sensing is performed on ultralong (mm range) high aspect ratio nanowall (50 nm width) surfaces functionalized with operator DNA tetO which is specifically bound by the sensor protein TetR. This sensor protein is released from the operator DNA in a dose dependent manner by exposing the device functionalized with this bound DNA-protein complex to tetracycline antibiotics. As a result, the electrical conductance is accordingly modulated by these surface net charge changes. The switching mechanism of sensor proteins attached at the functionalized surfaces and releasing them again by antibiotics is demonstrated. With the here presented device the detection limit is below the limits of prevailing detection methods. Moreover, the study is extended to detect antibiotic residues in spiked organic milk from cows far below the maximum residual level of the European Union. In spiked milk samples a detection limit for tetracycline concentrations in the 100 fM level was achieved. The nanowall devices are fabricated by atomic layer deposition-based spacer lithography on full wafer scale which is a simple approach capable for mass production. © 2013 The Royal Society of Chemistry.

Growth and differentiation of multicellular systems is orchestrated by spatially restricted gene expression programs in specialized subpopulations. The targeted manipulation of such processes by synthetic tools with high-spatiotemporal resolution could, therefore, enable a deepened understanding of developmental processes and open new opportunities in tissue engineering. Here, we describe the first red/far-red light-triggered gene switch for mammalian cells for achieving gene expression control in time and space. We show that the system can reversibly be toggled between stable on-and off-states using short light pulses at 660 or 740 nm. Red light-induced gene expression was shown to correlate with the applied photon number and was compatible with different mammalian cell lines, including human primary cells. The light-induced expression kinetics were quantitatively analyzed by a mathematical model. We apply the system for the spatially controlled engineering of angiogenesis in chicken embryos. The system's performance combined with cell-and tissue-compatible regulating red light will enable unprecedented spatiotemporally controlled molecular interventions in mammalian cells, tissues and organisms. © 2013 The Author(s).

The emergence and future of mammalian synthetic biology depends on technologies for orchestrating and custom tailoring complementary gene expression and signaling processes in a predictable manner. Here, we demonstrate for the first time multi-chromatic expression control in mammalian cells by differentially inducing up to three genes in a single cell culture in response to light of different wavelengths. To this end, we developed an ultraviolet B (UVB)-inducible expression system by designing a UVB-responsive split transcription factor based on the Arabidopsis thaliana UVB receptor UVR8 and the WD40 domain of COP1. The system allowed high (up to 800-fold) UVB-induced gene expression in human, monkey, hamster and mouse cells. Based on a quantitative model, we determined critical system parameters. By combining this UVB-responsive system with blue and red light-inducible gene control technology, we demonstrate multi-chromatic multi-gene control by differentially expressing three genes in a single cell culture in mammalian cells, and we apply this system for the multi-chromatic control of angiogenic signaling processes. This portfolio of optogenetic tools enables the design and implementation of synthetic biological networks showing unmatched spatiotemporal precision for future research and biomedical applications. © 2013 The Author(s) 2013. Published by Oxford University Press.

The chromophore 3-Z phycocyanobilin (PCB, (2R,3Z)-8,12-bis(2-carboxyethyl)-18-ethyl-3-ethylidene-2,7,13,17-tetramethyl-2,3-dihydrobilin-1,19(21H,24H)-dione) mediates red and far-red light perception in natural and synthetic biological systems. Here we describe a PCB synthesis strategy in mammalian cells. We optimize the production by co-localizing the biocatalysts to the substrate source, by coordinating the availability of the biocatalysts and by reducing the degradation of the reaction product. We show that the resulting PCB levels of 2 μM are sufficient to sustain the functionality of red light-responsive optogenetic tools suitable for the light-inducible control of gene expression in mammalian cells. © 2013 The Royal Society of Chemistry.

Light is fundamental to life on earth. Therefore, nature has evolved a multitude of photoreceptors that sense light across all kingdoms. This natural resource provides synthetic biology with a vast pool of light-sensing components with distinct spectral properties that can be harnessed to engineer novel optogenetic tools. These devices enable control over gene expression, cell morphology and signaling pathways with superior spatiotemporal resolution and are maturing towards elaborate applications in basic research, in the production of biopharmaceuticals and in biomedicine. This article provides a summary of the recent advances in optogenetics that use light for the precise control of biological functions in mammalian cells. © 2013 The Royal Society of Chemistry.

Time-resolved quantitative analysis of auxin-mediated processes in plant cells is as of yet limited. By applying a synergistic mammalian and plant synthetic biology approach, we have developed a novel ratiometric luminescent biosensor with wide applicability in the study of auxin metabolism, transport, and signalling. The sensitivity and kinetic properties of our genetically encoded biosensor open new perspectives for the analysis of highly complex auxin dynamics in plant growth and development.

Aluminium/aluminium oxide wires form under microgravity, earth conditions, and hypergravity in different forms. While under 0.04 G the biphasic wires are predominantly linear, they form bundles of wires of high curvature at 1 G and 1.8 G. The absence (0.04 G) and presence (1 G, 1.8 G) of gradients are reflected by the agglomeration and growth direction of the nanowires.

Alanes of the formula HxAlX3–x (x = 0, 1, 2, 3; X = Cl, Br, I) and the mixed halogen species HAlClBr were synthesized as N-methyl-piperidin (nmp) adducts HxAlX3–x·znmp (z = 1, 2) and HAlClBr·2nmp by different routes. The crystal and molecular structures of (H3Al·nmp)2, H2AlBr·2nmp, H2AlI·2nmp, the isotypic and isomorphous HAlBr2·2nmp, HAlI2·2nmp, as well as ACl3·nmp, ABr3·nmp, AI3·nmp, and HAlClBr·2nmp are described. The aluminum atoms in these molecular structures either form a pentagonal bipyramid as coordination sphere with the amine bases in apical positions or are in the center of a distorted tetrahedron (AlCl3·nmp, ABr3·nmp, AI3·nmp). In the nmp adduct of alane, a central Al2H2 ring is formed by dimerisation of AlH3, each aluminum atom displaying a further nmp ligand together with two terminal hydrogen atoms. The influence of the halogen atoms on the hydrogen bonds in the iso-structural compounds were studied by IR spectroscopy, showing in both series H2AlX·2nmp and HAlX2·2nmp (X = Cl, Br, I) a dependence of ν(Al–H) on the substitution pattern: 1770.2–1775.2–(not measured) cm–1 (H2AlX) and 1785.0–1788.0–1791.0 cm–1 (HAlX2). The δ values of the 27Al-NMR spectra in solution show dependences on the halide numbers bonded to aluminum and display a regular growth with heavier element in the H2AlX·2nmp series δ (27Al): 121.2 (Cl), 126.0 (Br), 131.5 (I)], whereas they have an unsteady evolution in the HAlX2·2nmp series [δ (27Al): 118.0 (Cl), 122.0 (Br), 108.0 (I)] and the AlX3·nmp series [δ (27Al): 102.1 (Cl), 104.8 (Br), 61.9 (I)].

The production of nano-calcium phosphate powders, such as HA (hydroxyapatite), from synthetic chemicals can be expensive and time consuming. The skeleton or shells of sea creatures (e.g. sea urchins, shells, corals) could be an alternative source of materials to produce very fine and even nano-structured calcium phosphate biomaterial powders. Ηydrothermal conversion under very high pressures or methods such as hot-plating (chemical) or ultrasonication (mechano-chemical), have been proposed to transform naturally derived CaCO3, e.g. aragonite, into apatite based materials. The aim of the present work was to prepare inexpensive nano-sized HA and TCP bioceramics powders from a local sea snail shells as a possible raw material for HA/TCP bioceramics. Empty shells of a local sea snail (Nassarius hinia reticulatus) from Marmara Sea, Turkey were collected from a beach near Istanbul. The collected shells were ground to a particle size 75μm. Thermal analyses (DTA/TGA) were performed to determine the exact CaCO3 content and thermal behavior. The raw powder was suspended in an aqueous media which was placed in an ultrasonic bath. The temperature was set at 80°C for 15min. Then, an equivalent (to CaO content) amount of H3PO4 was added drop by drop very gently into the solution. The reaction continued for 8h, following which the liquid component was evaporated off in an incubator at 100°C for 24h. The dried sediment was collected and heat treated at two different temperatures, 400 and 800°C. The morphology of the powders produced was examined using SEM. The crystalline phases were indentified using X-ray analysis.