Scientific publications

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.

In-stent restenosis is a common complication after stent surgery which leads to a dangerous wall narrowing of a blood vessel. Laser assisted patterning is one of the effective methods to modify the stent surface to control cell-surface interactions which play a major role in the restenosis. In this current study, 316LS stainless steel substrates are structured by focusing a femtosecond laser beam down to a spot size of 50 μm. By altering the laser induced spot density three distinct surfaces (low density (LD), medium density (MD) and high density (HD)) were prepared. While such surfaces are composed of primary microstructures, due to fast melting and re-solidification by ultra-short laser pulses, nanofeatures are also observed as secondary structures. Following a detailed surface characterization (chemical and physical properties of the surface), we used a well-established co-culture assay of human microvascular endothelial cells and human fibroblasts to check the cell compatibility of the prepared surfaces. The surfaces were analyzed in terms of cell adherence, proliferation, cell morphology and the differentiation of the fibroblast into the myofibroblast, which is a process indicating a general fibrotic shift within a certain tissue. It is observed that myofibroblast proliferation decreases significantly on laser treated samples in comparison to non-treated ones. On the other hand endothelial cell proliferation is not affected by the surface topography which is composed of micro- and nanostructures. Such surfaces may be used to modify stent surfaces for prevention or at least reduction of restenosis.

Modification of topography is an effective tool to control the cellular response such as cell function, adhesion and proliferation on surfaces. In this work, we present a novel method for fabrication of periodic surface structures by laser interference patterning (LIP) of biphasic Al/Al2O3 nanowires (NWs). These structures have a periodicity of 1-8 µm and a depth of 300-600 nm, depending on the incidence angle of the laser beam. Such hierarchical structures are composed of both micro- and nanofeatures. These periodic patterns lead to an alignment of glial cells and an axonal guidance from cultured dorsal root ganglia (DRG), along defined directions. The most pronounced alignment was seen at a periodicity of 2 µm.

Cell-material surface interaction plays a critical role in osseointegration of prosthetic implants used in orthopedic surgeries and dentistry. Different technical approaches exist to improve surface properties of such implants either by coating or by modification of their topography. Femtosecond laser treatment was used in this study to generate microspotted lines separated by 75, 125, or 175 μm wide nanostructured interlines on stainless steel (316L) plates. The hydrophobicity and carbon content of the metallic surface were improved simultaneously through this method. In vitro testing of the laser treated plates revealed a significant improvement in adhesion of human endothelial cells and human bone marrow mesenchymal stem cells (hBM MSCs), the cells involved in microvessel and bone formation, respectively, and a significant decrease in fibroblast adhesion, which is implicated in osteolysis and aseptic loosening of prostheses. The hBM MSCs showed an increased bone formation rate on the laser treated plates under osteogenic conditions; the highest mineral deposition was obtained on the surface with 125 μm interline distance (292 ± 18 mg/cm2 vs. 228 ± 43 mg/cm2 on untreated surface). Further in vivo testing of these laser treated surfaces in the native prosthetic implant niche would give a real insight into their effectiveness in improving osseointegration and their potential use in clinical applications.