Publikationen

Intercellular communication within an organism, between populations, or across species and kingdoms forms the basis of many ecosystems in which organisms coexist through symbiotic, parasitic, or predator-prey relationships. Using multistep airborne communication and signal transduction, we present synthetic ecosystems within a mammalian cell population, in mice, or across species and kingdoms. Inter- and intrakingdom communication was enabled by using sender cells that produce volatile aldehydes, small vitamin-derived molecules, or antibiotics that diffuse, by gas or liquid phase, to receiver cells and induce the expression of specific target genes. Intercellular and cross-kingdom communication was shown to enable quorum sensing between and among mammalian cells, bacteria, yeast and plants, resulting in precise spatiotemporal control of IFN-β production. Interconnection of bacterial, yeast, and mammalian cell signaling enabled the construction of multistep signal transduction and processing networks as well as the design of synthetic ecosystems that mimic fundamental coexistence patterns in nature, including symbiosis, parasitism, and oscillating predator-prey interactions. © 2007 by The National Academy of Sciences of the USA.

Bioprocess engineering has developed as a discipline to design optimal culture conditions and bioreactor operation protocols for production cell lines engineered for constitutive expression of desired protein pharmaceuticals. With the advent of heterologous gene regulation systems it has become possible to fine-tune expression of difficult-to-produce protein pharmaceuticals to optimal levels and to conditionally engineer cell metabolism for the best production performance. However, most of the small-molecules used to trigger expression of product or metabolic engineering product genes are incompatible with downstream processing regulations or process economics. Recent progress in product gene control design has resulted in the development of bioprocess-compatible regulation systems, which are responsive to physical parameters such as temperature or physiologic trigger molecules that are either an inherent part of host cell metabolism or intrinsic components of licensed protein-free cell culture media, such as redox status, vitamin H and gaseous acetaldehyde. While all of these systems have been shown to fine-tune product gene expression independent of the host cell metabolism some of them can be plugged into metabolic networks to capture critical physiologic parameters and convert them into an optimal production response. Assembly of individual product gene control modalities into synthetic networks has recently enabled construction of autonomously regulated time-delay or cell density-sensitive gene circuits, which trigger population-wide induction of product gene expression at a predefined time or culture density. We provide a comprehensive overview on the latest developments in the design of bioprocess-compatible product gene control systems. © 2007 Elsevier Ltd. All rights reserved.

Gene expression circuitries with time-delayed expression profiles regulate key events, such as oscillating systems, noise elimination, and coordinated multi-step processes, in all organisms from bacteria to mammalian cells. We present the rational synthesis of a genetic circuit displaying time-delayed expression in silico and in mammalian cells. The network is based on a time-delay circuit, where the tetracycline-responsive transactivator (tTA) induces expression of the pristinamycin-responsive repressor PIP-KRAB, which silences expression of the terminal human placental secreted alkaline phosphatase (SEAP). While the addition of pristinamycin I inactivates PIP-KRAB and results in the immediate resumption of SEAP expression, addition of tetracycline abolishes PIP-KRAB synthesis. Consequently, SEAP production remains repressed until the PIP-KRAB buffer in the cell is eliminated. We characterized in silico and in vivo the time-delayed expression properties and analyzed the impact of the size and stability of the PIP-KRAB buffer on fine-tuning of the response kinetics. This tunable time-delay circuitry represents a biologic building block for emulating a fundamental circuit topology in integrated artificial synthetic gene networks for the design of tailor-made cell types and organisms. © 2007 Wiley Periodicals, Inc.

Methods for specific immobilization, isolation and labeling of proteins are central to the elucidation of cellular functions. Based on bacterial repressor proteins, which bind to specific target sequences in response to small molecules (macrolide and tetracycline antibiotics) or environmental parameters (temperature), we have developed a set of protein tags (RepTAGs), which enable reversible immobilization of the protein of interest on a solid support for the isolation and quantification as well as for the specific labeling of target proteins with fluorescent dyes for tracking them within a complex protein mixture. Similarly, live mammalian cells were specifically labeled with a fluorescent operator sequence bound to RepTAGs, which were directed towards the cell surface for easy discrimination between transfected and untransfected cell populations. Based on the drug-responsive RepTAG-DNA interactions, it was also possible to quantify or discover antibiotics in environmental samples or compound libraries by means of rapid, sensitive detection methods involving fluorescence polarization and bioluminescence. We believe that the universally applicable RepTAGs will become essential for the analysis and manipulation of proteins in the most diverse areas of protein chemistry and cell biology. © 2007 Wiley Periodicals, Inc.

Time-delay circuitries in which a transcription factor processes independent input parameters can modulate NF-κB activation, manage quorum-sensing cross-talk, and control the circadian clock. We have constructed a synthetic mammalian gene network that processes four different input signals to control either immediate or time-delayed transcription of specific target genes. BirA-mediated ligation of biotin to a biotinylation signal-containing VP16 transactivation domain triggers heterodimerization of chimeric VP16 to a streptavidin-linked tetracycline represser (TetR). At increasing biotin concentrations up to 20 nM, TetR-specific promoters are gradually activated (off to on, input signal 1), are maximally induced at concentrations between 20 nM and 10 μM, and are adjustably shut off at biotin levels exceeding 10 μM (on to off, input signal 2). These specific expression characteristics with a discrete biotin concentration window emulate a biotin-triggered bandpass filter. Removal of biotin from the culture environment (input signal 3) results in time-delayed transgene expression until the intracellular biotinylated VP16 pool is degraded. Because the TetR component of the chimeric transactivator retains its tetracycline responsiveness, addition of this antibiotic (input signal 4) overrides biotin control and immediately shuts off target gene expression. Biotin-responsive immediate, bandpass filter, and time-delay transcription characteristics were predicted by a computational model and have been validated in standard cultivation settings or biopharmaceutical manufacturing scenarios using trangenic CHO-K1 cell derivatives and have been confirmed in mice. Synthetic gene circuitries provide insight into structure-function correlations of native signaling networks and foster advances in gene therapy and biopharmaceutical manufacturing. © 2007 by The National Academy of Sciences of the USA.

We describe an efficient inducible gene expression system in HEK.EBNA cells, a well-established cell system for the rapid transient expression of research-tool proteins. The transgene control system of choice is the novel acetaldehyde-inducible regulation (AIR) technology, which has been shown to modulate transgene levels following exposure of cells to acetaldehyde. For application in HEK.EBNA cells, AlcR transactivator plasmids were constructed and co-expressed with the secreted alkaline phosphatase (SEAP) gene under the control of a chimeric mammalian promoter (PAIR) for acetaldehyde-regulated expression. Several highly inducible transactivator cell lines were established. Adjustable transgene induction by gaseous acetaldehyde led to high induction levels and tight repression in transient expression trials and in stably transfected HEK.EBNA cell lines. Thus, the AIR technology can be used for inducible expression of any desired recombinant protein in HEK.EBNA cells. A possible application for inducible gene expression is a controlled proliferation strategy. Clonal HEK.EBNA cell lines, expressing the fungal transactivator protein AlcR, were engineered for gas-adjustable expression of the cell-cycle regulator p27Kip1. We show that expression of p27 Kip1 via transient or stable transfection led to a G1-phase specific growth arrest of HEK.EBNA cells. Furthermore, production pools engineered for gas-adjustable expression of p27Kip1 and constitutive expression of SEAP showed enhanced productive capacity. © 2006 Wiley Periodicals, Inc.

When Si8R*6 (R* = Si(tBu)3) is reacted with mild halogenation reagents like carbon tetrachloride, carbon tetrabromide, tin tetrachloride and iodine, new compounds are formed which besides the Si(tBu)3 groups have halide ligands and a silicon-silicon backbone which is either cyclic, bicyclic or polycyclic. We here report on the molecular and crystal structures of four derivatives: Si5R*3Cl5 (9), Si4R*3Br5 (10), Si8R*4Cl6 (11) and Si8R*3I11 (12). The molecule 9 has a bicyclic silicon back-bone with a three- and four-membered cycle sharing one edge, 10 is monocyclic with four membered silicon-silicon-ring, 11 is polycyclic and can be derived from a Si8-cube and 12 is bicyclic with a four- and five-membered silicon ring sharing one edge. We also propose some possible reaction mechanisms which follow from observations found during our experiments.

When the polycyclic alumosiloxane [Ph2SiO]8[AlO(OH)]4 is allowed to react with either cyclopentadienyl sodium in tetrahydrofuran or with dimethyl zinc in diethyl ether the organic ligands on the metal elements are eliminated as cyclopentadiene or methane and the metals are bonded to oxygen atoms in the alumosiloxane forming [Ph2SiO]8[AlO2(Na)]4∙5(THF) or [Ph2SiO]8[AlO(OH)]2[AlO2]2[Zn(OH)]2∙2(OEt2), respectively. X-ray structure determinations reveal that in the sodium derivative the original polycycle rests almost unchanged while in the zinc derivative the inner skeleton is rearranged.

Bis(hexamethyldisilazyl)tin(II) in excess reacts with the molecular alumosiloxane (Ph2SiO)8(AlO(OH))4 to yield the well known tin(II) derivative (Ph2SiO)8(AlO2)4Sn2 and the novel bicyclic [(Me3Si)2N]Sn[N(SiMe3)SiMe2CH2]2Sn[N(SiMe3)2] containing a tin-tin bond, which is formally related to the starting molecule by a missing hydrogen atom. From X-ray diffraction studies on single crystals it follows that the new compound has almost C2 point symmetry (the twofold axis intersecting the tin-tin bond) and that this tin-tin bond is the fusion edge of anellated fivefold Sn-Sn-N-Si-C rings. The bond connecting the almost tetrahedrally coordinated tin atoms (2.737(2) Å) is astonishingly very short. This feature is also reflected in the spectroscopic data (1H, 13C, 21Si, 119Sn-NMR) of the compound with a coupling constant 1J (117Sn/119Sn) of 9529 Hz.