Katja M. Arndt

Membrane‐active antimicrobial polymers (APs) are promising alternatives for treating infectious diseases as they are less susceptible to antimicrobial resistance development. However, their non‐specific cytotoxicity limits their clinical application. One interesting strategy to improve cytocompatibility is the incorporation of sugar units into APs. This approach takes advantage of specific interactions between sugars and protein receptors, such as lectins, on the surface of bacteria, potentially allowing for discrimination between different bacterial types. However, there is limited research on how sugar addition to cationic APs affects their selectivity and interactions with lectins. Herein, a library of acrylamide‐based cationic glycopolymers is synthesized by a xanthate‐supported photo‐initiated Reversible Additon‐Fragmentation chain Trasnfer (XPI‐RAFT) polymerization. Diverse sugars are added to this cationic scaffold by either chain extension with glycomonomers (based on fucose, glucose, mannose, and polyhydroxy acrylamide) or simply by reductive amination (lactose and melibiose). Various analytical techniques are used to probe the interaction of (glyco)polymers with bacteria, membrane models, and lectins. In general, (glyco)polyhydroxy incorporation proves to reduce the hydrophobicity of the cationic APs and consequently enhances hemocompatibility while maintaining the antimicrobial activity. Although sugar‐protein‐specific recognition may contribute to selectivity, the results demonstrate that electrostatic interaction predominantly drives the binding efficacy of cationic glycopolymers to bacterial membranes.

A method for fabricating anisotropic monodisperse silicon dioxide (SiO ₂ ) patchy microspheres is developed. In the protocol, we utilize a modified microcontact printing (µCP) technique, employing a polydimethylsiloxane (PDMS) elastomer with a regularly grooved surface topography as a stamp. The stamp's microscale channels create a confined environment for the SiO ₂ microspheres, as they match the particle dimensions (≈4 µm). The solid‐phase µCP routine allows for the transfer of functional trialkoxysilanes from the stamp to the particles exclusively at their contact faces. By applying a second stamp to the exposed side of the particles, a fourth patch with different chemical information is added, resulting in particles with four equidistant patches along the particle equator. This fabrication process, compatible with a manifold follow‐up chemistry, adds patches with adjustable characteristics. Consequently, a polymeric material containing the nucleobase adenine, is successfully added to the patches, which enables interactions with materials that possess its complementary base uracil. The method is suitable to fabricate particles possessing a C _4v and C _2v symmetry. Importantly, the protocol is easily adaptable to other particle dimensions and surface chemistry, possessing significant potential in the field of anisotropically functionalized spherical colloidal particles.

Devices sensing inputs and generating outputs are fundamental regulatory units, and as such are the basis of more complex regulatory networks. We provide an overview of regulatory devices used as fundamental regulatory building blocks in synthetic biology, and how complex genetic circuitry is being constructed from them. We first comprehensively explore devices operating at different levels of gene regulation, with action modes on the DNA sequence, to transcriptional, translational and post-translational control. We then discuss design principles of constructing genetic circuits from basic regulatory units, addressing challenges such as orthogonality, context-dependence, noise, and complexity. We present examples of genetic circuitry, including bistable switches, logic gates, signal amplification, memory devices and circuitry for biocomputation. How artificial genetic circuitry can be useful in real-life applications is illustrated with examples from bioproduction, living therapeutics, and biosafety. Our aim is to provide a comprehensive overview of the toolbox of regulatory devices and a profound understanding of their potential for constructing diverse genetic circuits and their applications.

We developed a method for fabricating anisotropic monodisperse silicon dioxide (SiO2) patchy microspheres. The protocol utilizes a modified microcontact printing (µCP) technique, employing a polydimethylsiloxane (PDMS) elastomer with a regularly grooved surface topography as a stamp. The stamp’s microscale channels create a confined environment for the SiO2 microspheres, as they match the particle dimensions (~4 µm). Our solid-phase µCP routine allows for the transfer of functional trialkoxysilanes from the elastomer stamp to the particles exclusively at their contact faces. By applying a second stamp to the exposed side of the particles, a fourth patch with different chemical information is added. This fabrication process, compatible with various follow-up chemistries, adds patches with adjustable characteristics. The method is suitable to fabricate particles possessing a C4v and C2v symmetry. Importantly, the protocol is easily adaptable to other particle dimensions and surface chemistries, indicating significant potential in the field of anisotropically functionalized spherical colloidal particles.

Water filtration is an important application to ensure the accessibility of clean drinking water. As requirements and contaminants vary on a local level, adjustable filter devices and their evaluation with contaminants are required. Within this work, modular filter devices are designed featuring an adjustable surface functionalization. For this purpose, 3D‐printed structures are created consisting of bio‐based poly(lactic acid) (PLA) that are manufactured by extrusion printing. The surface of PLA is activated with amino groups that are used to install xanthates as chain transfer agents. Subsequently, photo‐iniferter (PI) polymerization is used to create cationic polymer brushes on the surface of PLA substrates. Multiple surface characterization techniques are employed to prove successful growth of polymer brushes on PLA. After initial optimization studies on flat surfaces, filter devices are printed, functionalized, and used to remove bacteria from contaminated water. Significant reduction of the number of microorganisms is detected after filtration (single filtration or cycling) and contaminating organism can also be removed from freshwater samples by simple incubation with a 3D‐printed filter. The herein developed setup for producing functional filter devices and probing their performance in affinity filtration is a useful platform technology, enabling the rapid testing of polymer brushes for such applications.

Water filtration is an important application field to ensure the accessibility of clean drinking water. As requirements and contaminants vary on a local level, adjustable filter devices and their evaluation with contaminants are required. Within this work we design modular filter devices featuring an adjustable surface functionalization. For this purpose, we create 3D-printed structures fabricated of bio-based poly(lactic acid) (PLA) that are manufactured by extrusion printing. The surface of PLA is activated with amino groups that are used to install xanthates as chain transfer agents (CTA)s. Subsequently, photo-iniferter (PI)-RAFT polymerization is used to create cationic polymer brushes on the surface of PLA substrates. The polymerization is first optimized in solution and then transferred to the interface of 3D-printed devices. Multiple surface characterization techniques are employed to prove successful growth of polymer brushes on PLA. The degree of polymerization of cationic polymers is varied and found to influence the interaction of the interface with planktonic bacteria. After initial optimization studies on flat surfaces, filter devices are printed, functionalized, and used to remove bacteria from contaminated water. Significant reduction of microorganisms is detected after filtration (single filtration or cycling) and contaminating organism could also be removed from freshwater samples by simple incubation with a 3D-printed filter. The herein developed setup for producing functional filter devices and probing their performance in affinity filtration is a useful platform technology, enabling the implementation of polymer brushes for such applications. The combination of additive manufacturing and the modularity of the surface functionalization reaction enables rapid adjustability of the system.

Large quantities of the antibiotic florfenicol are used in animal farming and aquaculture, contaminating the ecosystem with antibiotic residues and promoting antimicrobial resistance, ultimately leading to untreatable multidrug-resistant pathogens. Florfenicol-resistant bacteria often activate export mechanisms that result in resistance to various structurally unrelated antibiotics. We devised novel strategies for the enzymatic inactivation of florfenicol in different media, such as saltwater or milk. Using a combinatorial approach and selection, we optimized a hydrolase (EstDL136) for florfenicol cleavage. Reaction kinetics were followed by time-resolved NMR spectroscopy. Importantly, the hydrolase remained active in different media, such as saltwater or cow milk. Various environmentally-friendly application strategies for florfenicol inactivation were developed using the optimized hydrolase. As a potential filter device for cost-effective treatment of waste milk or aquacultural wastewater, the hydrolase was immobilized on Ni-NTA agarose or silica as carrier materials. In two further application examples, the hydrolase was used as cell extract or encapsulated with a semi-permeable membrane. This facilitated, for example, florfenicol inactivation in whole milk, which can help to treat waste milk from medicated cows, to be fed to calves without the risk of inducing antibiotic resistance. Enzymatic inactivation of antibiotics, in general, enables therapeutic intervention without promoting antibiotic resistance.

Magnetic chains are of fundamental and technological interest. However, 1D assemblies of magnetic nanoparticles are only metastable such that their controlled organization requires the use of templates. Bacteriophages are human‐inoffensive viruses with a filamentous morphology that have been shown to exhibit great potential in materials research. Here, we thus utilized the M13 phage as a model for the formation of actuated magnetite nanoparticle superstructures. First, we built a sperm‐like ensemble by covalently attaching magnetic nanoparticles to the head of the phage. Second, chain‐like assemblies are obtained based on the electrostatic interactions between positively‐charged magnetite nanoparticles attached to the negatively‐charged phage surface. The nanoparticles‐phages assembly is steered by external magnetic fields. We anticipate such materials can find applications in nanotechnology or nanomedicine.

Recombinant adeno-associated viruses (rAAV) provide outstanding options for customization and superior capabilities for gene therapy. To access their full potential, facile genetic manipulation is pivotal, including capsid loop modifications. Therefore, we assessed capsid tolerance to modifications of the structural VP proteins in terms of stability and plasticity. Flexible glycine-serine linkers of increasing sizes were, at the genetic level, introduced into the 587 loop region of the VP proteins of serotype 2, the best studied AAV representative. Analyses of biological function and thermal stability with respect to genome release of viral particles revealed structural plasticity. In addition, insertion of the 29 kDa enzyme β-lactamase into the loop region was tested with a complete or a mosaic modification setting. For the mosaic approach, investigation of VP2 trans expression revealed that a Kozak sequence was required to prevent leaky scanning. Surprisingly, even the full capsid modification with β-lactamase allowed for the assembly of capsids with a concomitant increase in size. Enzyme activity assays revealed lactamase functionality for both rAAV variants, which demonstrates the structural robustness of this platform technology.

We report the design and characterization of photoactive yellow protein (PYP)-blue fluorescent protein (BFP)-fusion constructs that permit direct assay of reconstitution and function of the PYP domain. These constructs allow for in vivo testing of co-expression systems for enzymatic production of the p-coumaric acid-based PYP chromophore, via the action of tyrosine ammonia lyase (TAL) and p-coumaroyl-CoA ligase (pCL or 4CL). We find that different 4CL enzymes can function to reconstitute PYP including 4CL from Arabidopsis thaliana which can produce ~100% holo PYP protein under optimal conditions. BFP-fusion constructs additionally enable rapid analysis of effects of mutations on PYP photocycles. We use this BFP-fusion strategy to demonstrate in vivo reconstitution of several PYP-based optogenetic tools in Escherichia coli via biosynthesized chromophore, an important step for the use of these optogenetic tools in vivo in diverse hosts.