Biology subjects

Weber, W.

Publications and source records attributed to Weber, W..

At least 19 recordsLinked to original sources

bioRxiv2026

Data-driven predictive design of engineered living hydrogels

Engineered living materials (ELMs) offer a promising route to biologically manufactured materials for healthcare, construction and manufacturing. However, their rational design is limited by the lack of quantitative relationships linking design parameters to material properties. Here, we show that a tabular foundation model (TabPFN), informed by a small library of living hydrogels, accurately predicts macroscopic material properties from genetic and process parameters. Models were evaluated for predicting storage modulus (G'), fibre content, thickness, and permeability of Escherichia coli-produced living hydrogels containing CsgA-based fibres fused to genetically encoded PEG-like biopolymers. On an independent validation set, TabPFN achieved the strongest prediction for G' (R2 = 85.1%), reducing RMSE by 48.0% compared to linear regression. Property-guided design further enabled identification of parameters for achieving living hydrogels with desired properties. These results establish a broadly applicable framework for predictive design of ELMs, reducing experimental screening and accelerating the discovery of materials with targeted properties.

synthetic biology↗

bioRxiv2026

Tuning into xanthan: A conserved yet flexible polysaccharide utilization system in Microbacterium

Bacteria encounter structurally complex extracellular polysaccharides in natural environments, yet the regulatory and evolutionary basis of their utilization remains poorly understood. Here, we isolated a soil-derived Microbacterium strain, named Microbacterium xanthanicum UB-LE1, that grows on xanthan as the sole carbon source. We dissected the genetic and regulatory architecture underlying this capability. Genome sequencing combined with transcriptomic and proteomic profiling uncovered a discrete, strongly inducible regulon associated with xanthan utilization, encoding 23 proteins with five secreted proteins and three candidate transcriptional regulators. DNA-affinity purification sequencing confirmed two regulators binding to operons within the xanthan utilization locus. Comparative genomics across the Microbacteriaceae revealed conserved and lineage-specific features of this system and supports recent acquisition and modular integration of the locus, with at least two predominant architectural variants possibly shaped by substrate availability and ecological specialization. Coordinated induction at both the transcript and protein levels, together with two experimentally validated regulators, points to tight regulatory control of complex polysaccharide degradation in Microbacterium xanthanicum UB-LE1. Together, these findings provide mechanistic and evolutionary insight into how bacteria adapt to complex extracellular carbohydrates, expand current knowledge of xanthan turnover in microbial ecosystems, and establish a framework for exploring the emergence and diversification of specialized polysaccharide utilization pathways across bacterial taxa. IMPORTANCEMicroorganisms are central drivers of carbon turnover in soils and other terrestrial ecosystems, determining the availability of nutrients and shaping microbial community structure. A significant portion of soil carbon is contained in extracellular polysaccharides, yet the pathways by which microorganisms degrade these complex polymers remain poorly understood. Xanthan, a structurally complex and widely produced microbial exopolysaccharide, represents a persistent and largely overlooked carbon pool. By dissecting the genetic, regulatory, and evolutionary basis of xanthan utilization in Microbacterium xanthanicum UB-LE1, this study advances our understanding of how soil bacteria adapt to complex extracellular carbohydrates and how substrate availability shapes the emergence and diversification of specialized metabolic pathways. Importantly, the identification of additional xanthan-active enzymes and regulatory components in M. xanthanicum UB-LE1 opens opportunities for targeted modification of xanthan structure and properties, paving the way for new biotechnological applications in food, materials, and industrial biotechnology, while linking microbial ecology to functional innovation.

microbiology↗

bioRxiv2026

Genetically Programmed Shape-morphing of Engineered Living Materials

Engineered living materials (ELMs) promise genetically programmable functions by coupling biological regulation to synthetic material responses. Here, we introduce genetically encoded, reversible shape-morphing in a peptide-crosslinked polyethylene glycol (PEG) hydrogel whose network density is modulated by opposing enzymatic pairs that induce crosslinking or hydrolysis. This molecular programmability alternates the hydrogel between deswelling and swelling/disintegration and produces 2 - 5-fold changes in mechanical properties. By fabricating a bilayer hydrogel with an inert layer, these molecular modulations are translated into a reversible and directional motion with angular bending motions exceeding 80{degrees}. Further, by embedding genetically engineered bacteria or interfacing mammalian cells, producing the relevant enzymatic cues, the reversible shape-morphing of these ELMs is programmed at the genetic level. We further demonstrate genetically programmed, autonomous reversible bending in a bilayer hydrogel controlled by out-of-equilibrium counteracting biochemical reactions with dynamically changing respective reaction rates. This work establishes a concept where coordinated polymer/peptide material engineering and synthetic biology yield autonomous shape-morphing ELMs, opening avenues toward biohybrid soft robotics, adaptive microfluidic systems, and dynamic biomedical interfaces.

synthetic biology↗

bioRxiv2025

Single-domain antibodies targeting the pan-T-cell markers CD2 and CD7 as universal immunotherapy T-cell tracers

RationaleDespite significant advancements in immunotherapeutic approaches, such as immune checkpoint modulation and CAR-T cell therapy, reliable and universally applicable surrogate markers to monitor and evaluate the wide variety of therapeutic responses are still missing. This gap of information can lead to delayed assessment of responders versus non-responders and to misinterpretation, potentially resulting in premature or erroneous treatment decisions. Thus, a universal surrogate marker may be of preeminent value in monitoring immune responses, guiding immunotherapies, and ultimately improving patient outcomes. To address this challenge, we developed two immunoPET tracers based on single-domain antibodies (sdAbs) that target the pan T-cell markers CD2 and CD7 to monitor and visualize T cells distribution in vivo. MethodsThis study aimed to establish a comprehensive approach for clinical translation by assessing the feasibility and key characteristics of CD2- and CD7-sdAb for safe, effective clinical application. Binding specificity was confirmed through antigen transduction and knockout in tumor and T-cell leukemia cell lines. In vitro, cytokine secretion and T-cell cytotoxicity were evaluated, while a xenogeneic myeloid sarcoma mouse model was used to analyze T-cell cytotoxicity and visualize TCR-transduced CD8+ T cells at the tumor site using PET/MR imaging. ResultsThis study demonstrates the high specificity, affinity, and thermal stability of CD2- and CD7-sdAb, enabling efficient radiolabeling without impairing T-cell functionality. Specifically, binding to human CD8 T cells did not alter cytokine secretion or cytotoxicity in vitro. Furthermore, in vivo, the cytotoxic ability of transferred TCR-transduced human CD8+ T cells was not compromised upon i.v. application of CD2- and CD7-sdAb. Ultimately, intravenously injected 68Ga-NOTA-CD2-sdAb and 68Ga-NOTA-CD7-sdAb were respectively able to clearly depict and visualize previously administered TCR-transduced CD8+ T cells at the tumor site by PET/MRI. ConclusionsIn this study we have developed two pan T-cell tracers that are able to visualize human T cells and clearly differentiate between responding- and non-responding tumors while not impacting T-cell functionality in vitro and in vivo. The results provide a base for clinical use of these tracers as pan T-cell tracers applicable for any form of immunotherapy, making them a crucial tool in guiding immunotherapeutic decision-making.

immunology↗

bioRxiv2025

Genetically encoded SpyTag enables modular AAV retargeting via SpyCatcher-fused ligands for targeted gene delivery.

Recombinant adeno-associated viral (rAAV) vectors are a leading platform of in vivo gene therapy, valued for their excellent safety, broad serotype diversity, and scalable production. Targeted delivery through capsid display of ligands holds great promise, yet current retargeting strategies often rely on extensive capsid re-engineering and restrict the use of ligands incompatible with intracellular expression systems. Here, we present a modular AAV retargeting platform that, for the first time, employs the SpyTag/SpyCatcher system via genetic integration into the AAV2 capsid. SpyTag is a small peptide that forms a covalent, irreversible bond with its protein partner, SpyCatcher, allowing site-specific ligand coupling under physiological conditions. Inserting SpyTag into surface-exposed capsid sites enabled post-assembly functionalization of AAVs with SpyCatcher-fused targeting proteins. As proof-of-concept, we used SpyCatcher fusions with designed ankyrin repeat proteins (DARPins) specific for EGFR, EpCAM, and HER2. This conferred highly specific transduction of corresponding cancer cell lines with minimal off-target activity. Therapeutic potential was demonstrated by delivering a suicide gene, inducing selective cancer cell killing upon prodrug administration. This "one-fits-all" platform allows rapid and flexible retargeting without significantly altering the underlying vectors genome or production process. It supports the incorporation of large or complex ligands not amenable to genetic fusion and facilitates high-throughput preclinical evaluation strategies. By uniting capsid engineering with modular ligand display, our approach provides a scalable and versatile framework for precision gene delivery, broadening the applicability of rAAV in both therapeutic and discovery settings.

synthetic biology↗

bioRxiv2024

Bacterial Engineered Living Materials modulate Mechanosignaling in Mammalian Cells

Engineered living materials (ELMs) are gaining momentum for biomedical applications as self-replenishing drug depots, smart wound dressings, or as wearable sensors. Current studies on ELM-host interaction are mainly limited to the exchange of biochemical cues between ELMs and surrounding cells and tissues. Here we show that the genetically programmed mechanical properties of ELMs modulate mechanosignaling pathways in mammalian cells cultivated onto the living materials. To this aim, we genetically modulated curli fiber production in E. coli and analyzed the impact on the mechanical properties of the resulting ELMs. The living materials were used as matrix for the cultivation of mammalian cells engineered with a fluorescent reporter to indicate the activation of the mechano-responsive Hippo signaling pathway. We demonstrate that different genetically programmed ELM compositions translated into differential regulation of mechanosignaling in mammalian cells. These findings provide the perspective of using ELMs as extracellular matrix with genetically programmable mechanics for mammalian cells while also highlighting the need to consider the mechanical properties of therapeutic ELMs when assessing interaction with surrounding tissues. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/620857v1_ufig1.gif" ALT="Figure 1"> View larger version (68K): org.highwire.dtl.DTLVardef@1a1ef9borg.highwire.dtl.DTLVardef@79c49forg.highwire.dtl.DTLVardef@5bb04dorg.highwire.dtl.DTLVardef@193a808_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOToC figureC_FLOATNO C_FIG

bioengineering↗

bioRxiv2024

Influence of formaldehyde on signaling pathways when used in mammalian cell culture

Formaldehyde is the smallest existing aldehyde, a highly reactive color less gas at room temperature and ubiquitously present in our atmosphere. Because of its reactivity leading to the crosslinking of macromolecules like proteins, it is widely used in industrial applications, but also in cell biology in order to preserve cells and tissues for further analysis. In this work, we show that formaldehyde releasing solutions commonly used for fixation of cells, can diffuse via the gas phase to the neighboring well and influence signaling processes in the therein cultured cells. To analyze this effect, we utilized a stable reporter cell line for YAP signaling or a gene expression-based reporter for activation of the NF-{kappa}B pathway. Especially the stable reporter cell line can also be used as sensor for bioavailable formaldehyde. The observed impact of formaldehyde on cellular signaling underscores the need for careful planning of experimental protocols and emphasizes the importance of implementing proper controls when utilizing this reagent in cellular signaling analyses.

cell biology↗

bioRxiv2024

Metabolite-responsive Control of Transcription by Phase Separation-based Synthetic Organelles

Living natural materials have remarkable sensing abilities that translate external cues into functional changes of the material. The reconstruction of such sensing materials in bottom-up synthetic biology provides the opportunity to develop synthetic materials with life-like sensing and adaptation ability. Key to such functions are material modules that translate specific input signals into a biomolecular response. Here, we engineer a synthetic organelle based on liquid-liquid phase separation that translates a metabolic signal into the regulation of gene transcription. To this aim, we engineer the pyruvate-dependent repressor PdhR to undergo liquid-liquid phase separation in vitro by fusion to intrinsically disordered regions. We demonstrate that the resulting coacervates bind DNA harbouring PdhR-responsive operator sites in a pyruvate dose-dependent and reversible manner. We observed that the activity of transcription units on the DNA was strongly attenuated following recruitment to the coacervates. However, the addition of pyruvate resulted in a reversible and dose-dependent reconstitution of transcriptional activity. The coacervate-based synthetic organelles linking metabolic cues to transcriptional signals represent a materials approach to confer stimulus-responsiveness to minimal bottom-up synthetic biological systems and open opportunities in materials for sensor applications.

synthetic biology↗

bioRxiv2024

Activation of NF-κB signaling by optogenetic clustering of IKKα and β

A large percentage of proteins form higher-order structures in order to fulfill their function. These structures are crucial for the precise spatial and temporal regulation of the cellular signaling network. Investigation of this network requires sophisticated research tools, such as optogenetic tools, that allow dynamic control over the signaling molecules. Cryptochrome 2 and its variations are the best-characterized oligomerizing photoreceptors the optogenetics toolbox has to offer. Therefore, we utilized this switch and combined it with an eGFP-binding nanobody, to build a toolbox of optogenetic constructs that enables the oligomerization of any eGFP-tagged protein of interest. We further introduced the higher clustering variant Cry2olig and an intrinsically disordered region to create higher-order oligomers or phase-separated assemblies to investigate the impact of different oligomerization states on eGFP-tagged signaling molecules. We apply these constructs to cluster IKK and IKK{beta}, which resemble the central signaling integrator of the NF-{kappa}B pathway, thereby engineer a potent, blue-light-inducible activator of NF-{kappa}B signaling.

synthetic biology↗

bioRxiv2023

Engineering Material Properties of Transcription Factor Condensates to Control Gene Expression in Mammalian Cells and Mice

Phase separation of biomolecules into condensates is a key mechanism in the spatiotemporal organization of biochemical processes in cells. However, the impact of the material properties of biomolecular condensates on important processes, such as the control of gene expression, remains largely elusive. Here, we systematically tune the material properties of optogenetically induced transcription factor condensates and probe their impact on the activation of target promoters. We demonstrate that transcription factors in rather liquid condensates correlate with increased gene expression levels, whereas stiffer transcription factor condensates correlate with the opposite effect, a reduced activation of gene expression. We demonstrate the broad nature of these findings in mammalian cells and mice, as well as by using different synthetic and natural transcription factors. We observe these effects for both transgenic and cell-endogenous promoters. Our findings provide a novel materials-based layer in the control of gene expression, which opens novel opportunities in optogenetic engineering and synthetic biology.

synthetic biology↗

bioRxiv2023

The prion-like domain of the chloroplast RNA binding protein CP29A is required for cold-induced phase separation next to nucleoids and supports RNA splicing and translation during cold acclimation

Arabidopsis thaliana is capable of producing photosynthetic tissue with active chloroplasts at temperatures as low as 4{degrees}C, and this process depends on the presence of the nuclear-encoded, chloroplast-localized RNA-binding protein CP29A. In this study, we demonstrate that CP29A undergoes phase separation in vitro and in vivo in a temperature-dependent manner, which is mediated by a prion-like domain (PLD) located between the two RNA recognition motif (RRM) domains of CP29A. The resulting droplets display liquid-like properties and are found in close proximity to chloroplast nucleoids. The PLD is required to support chloroplast RNA splicing and translation in cold-treated tissue. Together, our findings suggest that plant chloroplast gene expression is compartmentalized by inducible condensation of CP29A at low temperatures, a mechanism that could play a crucial role for plant cold resistance.

plant biology↗

bioRxiv2023

Design of a Biohybrid Materials Circuit with Binary Decoder Functionality

Synthetic biology applies concepts from electrical engineering and information processing to endow cells with computational functionality. Transferring the underlying molecular components into materials and wiring them according to topologies inspired by electronic circuit boards has yielded materials systems that perform selected computational operations. However, the limited functionality of available building blocks is restricting the implementation of advanced information-processing circuits into materials. Here, we engineer a set of protease-based biohybrid modules the bioactivity of which can either be induced or inhibited. Guided by a quantitative mathematical model and following a design-build-test-learn cycle, we wire the modules according to circuit topologies inspired by electronic signal decoders, a fundamental motif in information processing. We design a 2-input/4-output binary decoder for the detection of two small molecules in a material framework that could perform regulated outputs in form of distinct protease activities. The here demonstrated smart material system is strongly modular and could be used for biomolecular information processing for example in advanced biosensing or drug delivery applications.

synthetic biology↗

bioRxiv2023

PenTag, a Versatile Platform for Covalent Bioconjugation, Purification, and Tagging of Proteins Towards the Development of Novel Biohybrid Material Systems

The site-specific and covalent conjugation of proteins on solid supports and in hydrogels is the basis for the synthesis of biohybrid materials offering broad applications. Current methods for conjugating proteins to desired targets are often challenging due to unspecific binding, unstable (non-covalent) coupling, or expensive and difficult-to-synthesize ligand molecules. Here, we present PenTag, an approach for the biorthogonal, highly-specific and covalent conjugation of a protein to its ligand for various applications in materials sciences. We engineered penicillin- binding protein 3 (PBP3) and showed that this protein can be used for the stable and spontaneous conjugation of proteins to dyes, polymers, or solid supports. We applied PenTag as a crosslinking tool for synthesizing stimuli-responsive hydrogels or for the development of a biohybrid material system performing computational operations emulating a 4:2 encoder. Based on this broad applicability and the use of a small, cheap and easy-to-functionalize ligand and a stable, soluble recombinant protein, we see PenTag as a versatile approach towards biohybrid material synthesis.

synthetic biology↗

bioRxiv2023

A photoreceptor-based hydrogel with red light-responsive reversible sol-gel transition as transient cellular matrix

Hydrogels with adjustable mechanical properties have been engineered as matrices for mammalian cells and allow the dynamic, mechano-responsive manipulation of cell fate and function. Recent research yielded hydrogels, where biological photoreceptors translated optical signals into a reversible and adjustable change in hydrogel mechanics. While their initial application provided important insights into mechanobiology, broader implementation is limited by a small dynamic range of addressable stiffness. Here, we overcome this limitation by developing a photoreceptor-based hydrogel with reversibly adjustable stiffness from 800 Pa to the sol state. The hydrogel is based on star-shaped polyethylene glycol, functionalized with the red/far-red light photoreceptor phytochrome B (PhyB), or phytochrome-interacting factor 6 (PIF6). Upon illumination with red light, PhyB heterodimerizes with PIF6, thus crosslinking the polymers and resulting in gelation. However, upon illumination with far-red light, the proteins dissociate and trigger a complete gel-to-sol transition. We comprehensively characterize the hydrogels light-responsive mechanical properties and apply it as reversible extracellular matrix for the spatiotemporally controlled deposition of mammalian cells within a microfluidic chip. We anticipate that this technology will open new avenues for the site- and time-specific positioning of cells and will contribute to overcome spatial restrictions.

synthetic biology↗

bioRxiv2023

Engineering a material-genetic interface as safety switch for embedded therapeutic cells

Encapsulated cell-based therapies involve the use of genetically-modified cells embedded in a material in order to produce a therapeutic agent in a specific location in the patients body. This approach has shown great potential in animal model systems for treating diseases such as type I diabetes or cancer, with selected approaches having been tested in clinical trials. Despite the promise shown by encapsulated cell therapy, though, there are safety concerns yet to be addressed, such as the escape of the engineered cells from the encapsulation material and the resulting production of therapeutic agents at uncontrolled sites of the body. For that reason, there is great interest in the implementation of safety switches that protect from those side effects. Here, we develop a material-genetic interface as safety switch for engineered mammalian cells embedded into hydrogels. Our switch allows the therapeutic cells to sense whether they are embedded in the hydrogel by means of a synthetic receptor and signaling cascade that link transgene expression to the presence of an intact embedding material. The system design is highly modular, allowing its flexible adaptation to other cell types and embedding materials. This autonomously acting switch constitutes an advantage over previously described safety switches, which rely on user-triggered signals to modulate activity or survival of the implanted cells. We envision that the concept developed here will advance the safety of cell therapies and facilitate their translation to clinical evaluation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/535359v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@15e5005org.highwire.dtl.DTLVardef@d793a2org.highwire.dtl.DTLVardef@11d92f6org.highwire.dtl.DTLVardef@9b8128_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Real-time monitoring of cell surface protein arrival with split luciferases

Each cell in a multicellular organism permanently adjusts the concentration of its cell surface proteins. In particular, epithelial cells tightly control the number of carriers, transporters and cell adhesion proteins at their plasma membrane. However, the sensitively measuring the cell surface concentration of a particular protein of interest in live cells and in real time represents a considerable challenge. Here, we introduce a novel approach based on split luciferases, which uses one luciferase fragment as tag on the protein of interest and the second fragment as supplement to the extracellular medium. Once the protein of interest arrives at the cell surface, the luciferase fragments complement and generate luminescence. We compared the performance of split Gaussia luciferase and split Nanoluciferase by using a system to synchronize biosynthetic trafficking with conditional aggregation domains. The best results were achieved with split Nanoluciferase, for which luminescence increased more than 6000-fold upon recombination. Furthermore, we showed that our approach can separately detect and quantify the arrival of membrane proteins at the apical and basolateral plasma membrane in single polarized epithelial cells by detecting the luminescence signals with a microscope, thus opening novel avenues for characterizing the variations in trafficking in individual epithelial cells. SynopsisWe present here a novel method to determine the arrival of a membrane protein of interest at the cell surface in real time and with sufficient sensitivity to achieve single-cell resolution. This allows measuring detailed kinetics of plasma membrane trafficking and thus to detect also minor changes in protein sorting and intracellular trafficking. Furthermore, the single-cell sensitivity of the method will enable to systematically characterize variations in trafficking between neighboring cells within a multicellular organism.

cell biology↗

bioRxiv2023

SPI-1 virulence gene expression modulates motility of Salmonella Typhimurium in a proton motive force- and adhesins-dependent manner

Both the bacterial flagellum and the evolutionary related injectisome encoded on the Salmonella pathogenicity island 1 (SPI-1) play crucial roles during the infection cycle of Salmonella species. The interplay of both is highlighted by the complex cross-regulation that includes transcriptional control of the flagellar master regulatory operon flhDC by HilD, the master regulator of SPI-1 gene expression. Contrary to the HilD-dependent activation of flagellar gene expression, we report here that activation of HilD resulted in a dramatic loss of motility, which was dependent on the presence of SPI-1. Single cell analyses revealed that HilD-activation results in a SPI-1-dependent induction of the stringent response and a pronounced decrease of proton motive force (PMF), while flagellation was not affected. We further found that activation of HilD enhanced the adhesion of Salmonella to epithelial cells. A transcriptome analysis revealed a concomitant upregulation of several adhesin systems, which when overproduced, phenocopied the HilD-induced motility defect. We propose that a combination of SPI-1-dependent depletion of the PMF and upregulation of adhesins upon HilD-activation allows flagellated Salmonella to rapidly modulate their motility during infection, thereby enabling efficient adhesion to host cells and delivery of effector proteins.

microbiology↗

bioRxiv2022

Systematic Evaluation of Self-Assembling Membrane Peptides to Form Nanopores in the context of the Functional Nanopore Screen

The functional analysis of protein nanopores is typically conducted in planar lipid bilayers or liposomes exploiting high-resolution but low-throughput electrical and optical read-outs. Yet, the reconstitution of protein nanopores in vitro still constitutes an empiric and low-throughput process. Addressing these limitations, nanopores can now be analyzed using the functional nanopore (FuN) screen exploiting genetically-encoded fluorescent protein sensors that resolve distinct nanopore-dependent Ca2+ in- and efflux patterns across the inner membrane of Escherichia coli. With a primary proof-of-concept established for the S2168 holin, and thereof based recombinant nanopore assemblies, the question arises to what extent alternative nanopores can be analyzed with the FuN screen and to what extent alternative fluorescent protein sensors can be adapted. Focussing on self-assembling membrane peptides, three sets of 13 different nanopores are assessed for their capacity to form nanopores in the context of the FuN screen. Nanopores tested comprise both natural and computationally-designed nanopores. Further, the FuN screen is extended to K+-specific fluorescent protein sensors to provide a complementary read-out to Ca2+. Finally, a comparison with high-resolution biophysical and electrophysiological studies in planar lipid bilayers provides an experimental benchmark for future studies.

synthetic biology↗