Publikationen

Methodologies for the controlled delivery of genetic information into target cells are of utmost importance for genetic engineering in both fundamental and applied research. However, available methods for efficient gene transfer into user-selected or even single cells suffer from low throughput, the need for complicated equipment, high invasiveness, or side effects by off-target viral uptake. Here, we engineer an adeno-associated viral (AAV) vector system that transfers genetic information into native target cells upon illumination with cell-compatible red light. This OptoAAV system allows adjustable and spatially resolved gene transfer down to single-cell resolution and is compatible with different cell lines and primary cells. Moreover, the sequential application of multiple OptoAAVs enables spatially resolved transduction with different transgenes. The approach presented is likely extendable to other classes of viral vectors and is expected to foster advances in basic and applied genetic research. Copyright © 2021 The Authors, some rights reserved.

The quest to engineer increasingly complex synthetic gene networks in mammalian and plant cells requires an ever-growing portfolio of orthogonal gene expression systems. To control gene expression, light is of particular interest due to high spatial and temporal resolution, ease of dosage and simplicity of administration, enabling increasingly sophisticated man–machine interfaces. However, the majority of applied optogenetic switches are crowded in the UVB, blue and red/far-red light parts of the optical spectrum, limiting the number of simultaneously applicable stimuli. This problem is even more pertinent in plant cells, in which UV-A/B, blue, and red light-responsive photoreceptors are already expressed endogenously. To alleviate these challenges, we developed a green light responsive gene switch, based on the light-sensitive bacterial transcription factor CarH from Thermus thermophilus and its cognate DNA operator sequence CarO. The switch is characterized by high reversibility, high transgene expression levels, and low leakiness, leading to up to 350-fold induction ratios in mammalian cells. In this chapter, we describe the essential steps to build functional components of the green light-regulated gene switch, followed by detailed protocols to quantify transgene expression over time in mammalian cells. In addition, we expand this protocol with a description of how the optogenetic switch can be implemented in protoplasts of A. thaliana. © 2021, Springer Science+Business Media, LLC, part of Springer Nature.

Three recent approvals and over 100 ongoing clinical trials make adeno-associated virus (AAV)-based vectors the leading gene delivery vehicles in gene therapy. Pharmaceutical companies are investing in this small and nonpathogenic gene shuttle to increase the therapeutic portfolios within the coming years. This prospect of marking a new era in gene therapy has fostered both investigations of the fundamental AAV biology as well as engineering studies to enhance delivery vehicles. Driven by the high clinical potential, a new generation of synthetic-biologically engineered AAV vectors is on the rise. Concepts from synthetic biology enable the control and fine-tuning of vector function at different stages of cellular transduction and gene expression. It is anticipated that the emerging field of synthetic-biologically engineered AAV vectors can shape future gene therapeutic approaches and thus the design of tomorrow's gene delivery vectors. This review describes and discusses the recent trends in capsid and vector genome engineering, with particular emphasis on synthetic-biological approaches. © 2021 The Authors. Advanced Science published by Wiley-VCH GmbH

Light-inducible gene switches represent a key strategy for the precise manipulation of cellular events in fundamental and applied research. However, the performance of widely used gene switches is limited due to low tissue penetrance and possible phototoxicity of the light stimulus. To overcome these limitations, we engineer optogenetic synthetic transcription factors to undergo liquid-liquid phase separation in close spatial proximity to promoters. Phase separation of constitutive and optogenetic synthetic transcription factors was achieved by incorporation of intrinsically disordered regions. Supported by a quantitative mathematical model, we demonstrate that engineered transcription factor droplets form at target promoters and increase gene expression up to fivefold. This increase in performance was observed in multiple mammalian cells lines as well as in mice following in situ transfection. The results of this work suggest that the introduction of intrinsically disordered domains is a simple yet effective means to boost synthetic transcription factor activity. Copyright © 2021 The Authors, some rights reserved.

Activation of T cells by agonistic peptide-MHC can be inhibited by antagonistic ones. However, the exact mechanism remains elusive. We used Jurkat cells expressing two different TCRs and tested whether stimulation of the endogenous TCR by agonistic anti-Vβ8 antibodies can be modulated by ligand-binding to the second, optogenetic TCR. The latter TCR uses phytochrome B tetramers (PhyBt) as ligand, the binding half-life of which can be altered by light. We show that this half-life determined whether the PhyBt acted as a second agonist (long half-life), an antagonist (short half-life) or did not have any influence (very short half-life) on calcium influx. A mathematical model of this cross-antagonism shows that a mechanism based on an inhibitory signal generated by early recruitment of a phosphatase and an activating signal by later recruitment of a kinase explains the data. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

The conquest of fire has been an essential component of the civilizing process. Milazzo and Buehler from MIT spearhead a novel direction of research by seeking inspiration from fire through deep learning, paving a new avenue for the design and fabrication of novel materials.

Here, fundamental aspects affecting template-assisted engineering of oxidase-associated peroxide oxidation co-catalysis of the modeled microanalytical system based on the hybrid palladium nanoparticles (Pd-NPs) with tailored functional properties were studied. By an accurate tuning and validation of the experimental setup, a modular Pd-NPs-doped one-pot/one-electrode amperometric nanobiosensor for advanced multiplex analyte detection was constructed. The specific operational conditions (electrochemical read-out mode, pH, regeneration procedure) of the modular one-pot/one-electrode nanobiosensor allowed a reliable sensing of L-lactate (with linear dynamic range, LDR=500 µM – 2 mM, R2 =0.977), D-glucose (with LDR=200 µM – 50 mM, R2 =0.987), hydrogen peroxide (with LDR=20 µM – 100 mM, R2 =0.998) and glutaraldehyde (with LDR=1 – 100 mM, R2 =0.971). In addition, mechanistic aspects influencing the performance of Pd-NPs-doped one-pot/one-electrode for multiplex analyte sensing were studied in detail. The designed one-pot/one-electrode amperometric nanobiosensor showed a thin layer electrochemical behavior that greatly enhanced electron transfer between the functional hybrid layer and the electrode. Finally, a specific regeneration procedure of the hybrid one-pot/one-electrode and algorithm towards its usage for modular biocatalysis were developed. The reported strategy can readily be considered as a guideline towards the fabrication of commercialized nanobiosensors with tailored properties for advanced modular biocatalysis.

Abstract In the presence of water and amines the etherate of bicyclic Al2(OSiPh2OSiPh2O)3 (II a) can be used to generate novel alumosiloxane polycycles like [O(Ph2SiOSiPh2)O−]2Al2O[O(Ph2SiOSiPh2)O] ⋅ 2 H2N+Et2 (1), [O(Ph2SiOSiPh2)O−]2Al2[O(Ph2Si)O]2 ⋅ 2 HN+Et3 (2), [O(Ph2SiOSiPh2)O−]2Al2[O(Ph2SiOSiPh2)O]2 ⋅ 2 HN+Et3 (3 a, 3 b), which crystallizes in two different phases, and [O(Ph2SiOSiPh2)O−]2Al2[O(Ph2SiOSiPh2)O]2 ⋅ 2 HN+(CH2CH2)3N (4). As a common structural feature of these compounds two aluminum atoms which are incorporated in six-membered Al[O(SiPh2OSiPh2)O−] rings are connected as spiro cyclic centers through oxygen and/or siloxane bridges [(OSiPh2)nO] (n=1, 2) to form an assembly of three fused rings at the aluminum corners. The central ring is either eight- (1, 2) or twelve-membered (3, 4). Alkyl ammonium cations balance the charges and form hydrogen bridges to oxygen atoms of the six membered rings. The pentacyclic (Ph2SiO)8[Al(O)OH]4 (I) can be used indirectly (addition of water) and directly as chelating ligand versus Co(II)Cl and In-CH3 fragments as shown with the isolated and structurally characterized compounds (HN+Et3)2{[(Ph2Si)2O3][Al4(OH)4O2](CoCl)2}2− (5 a, 5 b) and (Ph2SiO)8[AlO(OH)]2[AlO2]2(InCH3) ⋅ 2 O(CH2)4 (6).

Hydrogels are widely used as hydrated matrices for cell encapsulation in a number of applications, spanning from advanced 3D cultures and tissue models to cell-based therapeutics and tissue engineering. Hydrogel formation in the presence of living cells requires cross-linking reactions that proceed efficiently under close to physiological conditions. Recently, the nucleophilic aromatic substitution of phenyl-oxadiazole (Ox) methylsulfones (MS) by thiols was introduced as a new cross-linking reaction for cell encapsulation. Reported poly(ethylene glycol) (PEG)-based hydrogels featured tunable gelation times within seconds to a few minutes within pH 8.0 to 6.6 and allowed reasonably good mixing with cells. However, their rapid degradation prevented cell cultures to be maintained beyond 1 week. In this Article, we present the reactivity optimization of the heteroaromatic ring of the MS partner to slow down the cross-linking kinetics and the degradability of the derived hydrogels. New MS substrates based on phenyl-tetrazole (Tz) and benzothiazole (Bt) rings, with lower electrophilicity than Ox, were synthesized by simple pathways. When mixed with PEG-thiol, the novel PEG-MS extended the working time of precursor mixtures and allowed longer term cell culture. The Tz-based MS substrate was identified as the best candidate, as it is accessible by simple chemical reactions from cost-effective reactants, hydrogel precursors show good stability in aqueous solution and keep high chemoselectivity for thiols, and the derived Tz gels support cell cultures for >2 weeks. The Tz system also shows tunable gelation kinetics within seconds to hours and allows comfortable manipulation and cell encapsulation. Our findings expand the toolkit of thiol-mediated chemistry for the synthesis of hydrogels with improved properties for laboratory handling and future automatization.

Faradaic electrode materials have significantly improved the performance of membrane capacitive deionization, which offers an opportunity to produce freshwater from seawater or brackish water in an energy-efficient way. However, Faradaic materials hold the drawbacks of slow desalination rate due to the intrinsic low ion diffusion kinetics and inferior stability arising from the volume expansion during ion intercalation, impeding the engineering application of capacitive deionization. Herein, a pseudocapacitive material with hollow architecture was prepared via template-etching method, namely, cuboid cobalt hydroxide, with fast desalination rate (3.3 mg (NaCl)·g-1 (h-Co(OH)2)·min-1 at 100 mA·g-1) and outstanding stability (90% capacity retention after 100 cycles). The hollow structure enables swift ion transport inside the material and keeps the electrode intact by alleviating the stress induced from volume expansion during the ion capture process, which is corroborated well by in situ electrochemical dilatometry and finite element simulation. Additionally, benefiting from the elimination of unreacted bulk material and vertical cobalt hydroxide nanosheets on the exterior surface, the synthesized material provides a high desalination capacity ( mg (NaCl)·g-1 (h-Co(OH)2) at 30 mA·g-1). This work provides a new strategy, constructing microscale hollow faradic configuration, to further boost the desalination performance of Faradaic materials.