INMdok (Leibniz Institute for New Materials)
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Expanding the genetic programmability of Lactiplantibacillus plantarum
Get PDFLactobacilli are ubiquitous in nature and symbiotically provide health benefits for countless organisms including humans, animals and plants. They are vital for the fermented food industry and are being extensively explored for healthcare applications. For all these reasons, there is considerable interest in enhancing and controlling their capabilities through the engineering of genetic modules and circuits. One of the most robust and reliable microbial chassis for these synthetic biology applications is the widely used Lactiplantibacillus plantarum species. However, the genetic toolkit needed to advance its applicability remains poorly equipped. This mini-review highlights the genetic parts that have been discovered to achieve food-grade recombinant protein production and speculates on lessons learned from these studies for L. plantarum engineering. Furthermore, strategies to identify, create and optimize genetic parts for real-time regulation of gene expression and enhancement of biosafety are also suggested
Targeting Intracellular Bacteria with Dual Drug-loaded Lactoferrin Nanoparticles
Get PDFTreatment of microbial infections is becoming daunting because of widespread antimicrobial resistance. The treatment challenge is further exacerbated by the fact that certain infectious bacteria invade and localize within host cells, protecting the bacteria from antimicrobial treatments and the host’s immune response. To survive in the intracellular niche, such bacteria deploy surface receptors similar to host cell receptors to sequester iron, an essential nutrient for their virulence, from host iron-binding proteins, in particular lactoferrin and transferrin. In this context, we aimed to target lactoferrin receptors expressed by macrophages and bacteria; as such, we prepared and characterized lactoferrin nanoparticles (Lf-NPs) loaded with a dual drug combination of antimicrobial natural alkaloids, berberine or sanguinarine, with vancomycin or imipenem. We observed increased uptake of drug-loaded Lf-NPs by differentiated THP-1 cells with up to 90% proportion of fluorescent cells, which decreased to about 60% in the presence of free lactoferrin, demonstrating the targeting ability of Lf-NPs. The encapsulated antibiotic drug cocktail efficiently cleared intracellular Staphylococcus aureus (Newman strain) compared to the free drug combinations. However, the encapsulated drugs and the free drugs alike exhibited a bacteriostatic effect against the hard-to-treat Mycobacterium abscessus (smooth variant). In conclusion, the results of this study demonstrate the potential of lactoferrin nanoparticles for the targeted delivery of antibiotic drug cocktails for the treatment of intracellular bacteria
Mechanistic aspects of glycerol oxidation on palladium electrocatalysts in model aqueous and fermentation media solutions
Get PDFHerein, a study dealing with a progress on palladium (Pd) electrocatalysts for an efficient glycerol electrooxidation in model aqueous and real fermentation solutions with special focus on some physicochemical parameters (e.g., the impact of adsorption stage of multiple species, presence of oxygen, influence of anodic limits and Pd-size) was conducted. During the course of investigations by tandem of an optical oxygen minisensor and cyclic voltammetry a significant impact of oxygen on the efficiency of glycerol electrooxidation on Pd electrocatalysts at alkaline pH in model aqueous and yeast fermentation media was revealed. The obtained knowledge was used for the optimization of an assay utilizing Pd-sensing layers for glycerol determination and quantification in yeast fermentation medium. Received results showed a satisfactory agreement with a control measurement carried out by gas chromatography mass-spectrometry
Self-Lubricating, Living Contact Lenses
Get PDFThe increasing prevalence of dry eye syndrome in aging and digital societies compromises long-term contact lens (CL) wear and forces users to regular eye drop instillation to alleviate discomfort. Here a novel approach with the potential to improve and extend the lubrication properties of CLs is presented. This is achieved by embedding lubricant-secreting biofactories within the CL material. The self-replenishable reservoirs autonomously produce and release hyaluronic acid (HA), a natural lubrication and wetting agent, long term. The hydrogel matrix regulates the growth of the biofactories and the HA production, and allows the diffusion of nutrients and HA for at least 3 weeks. The continuous release of HA sustainably reduces the friction coefficient of the CL surface. A self-lubricating CL prototype is presented, where the functional biofactories are contained in a functional ring at the lens periphery, outside of the vision area. The device is cytocompatible and fulfils physicochemical requirements of commercial CLs. The fabrication process is compatible with current manufacturing processes of CLs for vision correction. It is envisioned that the durable-by-design approach in living CL could enable long-term wear comfort for CL users and minimize the need for lubricating eye drops
Polyphosphonate covalent organic frameworks
Get PDFHerein, we report polyphosphonate covalent organic frameworks (COFs) constructed via P-O-P linkages. The materials are synthesized via a single-step condensation reaction of the charge-assisted hydrogen-bonded organic framework, which is constructed from phenylphosphonic acid and 5,10,15,20‐tetrakis[p‐phenylphosphonic acid]porphyrin and is formed by simply heating its hydrogen-bonded precursor without using chemical reagents. Above 210 °C, it becomes an amorphous microporous polymeric structure due to the oligomerization of P-O-P bonds, which could be shown by constant-time solid-state double-quantum 31P nuclear magnetic resonance experiments. The polyphosphonate COF exhibits good water and water vapor stability during the gas sorption measurements, and electrochemical stability in 0.5 M Na2SO4 electrolyte in water. The reported family of COFs fills a significant gap in the literature by providing stable microporous COFs suitable for use in water and electrolytes. Additionally, we provide a sustainable synthesis route for the COF synthesis. The narrow pores of the COF effectively capture CO2
Amine-containing block copolymers for efficient catalyst-free hydroamination and preparation of functional metallopolymers
Get PDFCobaltocenium-containing polymers, an emerging class of materials, have historically been challenging to prepare due to their chemical robustness. In this work, we introduce a novel and highly efficient method for their preparation based on methacrylate-containing block copolymers (BCPs), allowing segment-selective introduction of functional moieties. The catalyst-free and quantitative hydroamination reaction we introduce has proven successful for the post-modification of amine-containing polymers with cobaltocenium. To demonstrate the versatility of this method, we successfully synthesized a series of BCPs consisting of polystyrene and a 5 to 20 wt% poly(tert-butyl aminoethyl methacrylate) (PtBAEMA) segment by living anionic polymerization. The selective functionalization with ethynyl-cobaltocenium hexafluorophosphate results in adjustable 5 to 40 wt% cobaltocenium units in the polymer as part of the PtBAEMA block segment. The success was monitored by IR spectroscopy, and the quantitative incorporation of the cobaltocenium moiety was verified by 1H NMR, UV-Vis spectroscopy, and TGA. DSC proved the block-selective cobaltocenium introduction by an additional glass transition temperature at 154 °C, and the strong microphase separation character of the amphiphilic BCPs leads to lamellar structures in the bulk state, as proven by TEM investigations. Finally, the water contact angle on polymer films is compared, showing polarity inversion and tunability upon conversion of hydrophilic amine to hydrophobic cobaltocenium hexafluorophosphate moieties. This successful synthesis and characterization of cobaltocenium-containing BCPs not only paves the way for a new class of metallopolymers but also offers functionalization possibilities for a variety of other responsive moieties, providing access to functional BCPs
A comparative Study between Thiol-ene and Acrylate Photocrosslinkable Hyaluronic Acid Hydrogel inks for Digital Light Processing
Get PDFPhotopolymerizable formulations based on the thiol-ene crosslinking are considered better alternatives than methacrylated counterparts for light-based fabrication processes. The thiol-ene photoreaction requires lower light doses for polymerization and leads to networks with lower molecular heterogeneity. For hydrogels at low polymer concentrations as used in bioinks (<8 wt%), faster polymerization rates and higher conversion of the crosslinking reaction have been reported in thiol-ene systems. Here we quantify further differences between thiol-ene and methacrylated crosslinked hydrogels for HA functionalized with norbornene, allyl ether or methacrylate groups. We show storage stability of the thiol-ene hydrogel precursors over 15 months, 3.8 times higher than the methacrylated derivative. Photorheology experiments demonstrated up to 4.7-times faster photocrosslinking. Network formation in photoinitiated thiol-ene HA crosslinking allows higher temporal control than in methacrylated HA, which shows long post-illumination hardening. Using digital light processing, 4% w/v HA hydrogels polymerized with a dithiol crosslinker allowed printing of 13.5x4x1 mm3 layers with holes of 100 µm resolution within 2 s. Considering that ene-derivatized hydrogel precursors are less commercially available than methacrylated counterparts, our results are important to estimate the extent to which the synthetic effort of introducing –ene functions can pay off in the printing step
Uric acid detection by hydrogen peroxide independent biosensors: Novel insights and applications
Get PDFUric acid (UA) is one of the most electroactive low molecular weight compounds that can be electrochemically oxidized on the surfaces of numerous noble and non-noble electrocatalysts under applied polarization. Consequently, enzymatic determination of UA in model and real samples is complicated by possible interference between electrochemical and biochemical routes. Herein, a novel strategy for amperometric enzymatic hydrogen peroxide independent UA sensing at low concentrations (e.g., below 50 µM) is proposed. The UA-sensitive strategy relies on the use of screen printed electrodes modified by an electrodeposited hybrid functional sensing film comprising a non-noble electrocatalyst, a bioorganic layer containing enzyme uricase (UOx), and data acquisition enabling the biochemical transformation of UA to be distinguished from the electrochemical oxidation route. Performed selectivity test utilizing adenine, xanthine, urea, ascorbic acid, ethanol and glycerol did not reveal interferences during detection of UA. This proposed approach was tested for UA detection in model and fermentation samples. The quantitative results obtained in fermentation samples were validated through optical oxygen mini sensor studies and fluorescence-based bioassays
Mechanically Stable, Binder-Free, and Free-Standing Vanadium Trioxide/Carbon Hybrid Fiber Electrodes for Lithium-Ion Batteries
Get PDFBinder is a crucial component in present-day battery electrodes but commonly contains fluorine and requires coating processing using organic (often toxic) solvents. Preparing binder-free electrodes is an attractive strategy to make battery electrode production and its end-of-use waste greener and safer. Herein, electrospinning is employed to prepare binder-free and self-standing electrodes. Such electrodes often suffer from low flexibility, and the correlation between performance and flexibility is usually overlooked. Processing parameters affect the mechanical properties of the electrodes, and for the first time it is reported that mechanical flexibility directly influences the electrochemical performance of the electrode. The importance is highlighted when processing parameters advantageous to powder materials, such as a higher heat treatment temperature, harm self-standing electrodes due to deterioration of fiber flexibility. Other strategies, such as conductive carbon addition, can be employed to improve the cell performance, but their effect on the mechanical properties of the electrodes must be considered. Rapid heat treatment achieves self-standing V2O3 with a capacity of 250 mAh g−1 at 250 mA g−1 and 390 mAh g−1 at 10 mA g−
In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1
Get PDFLactobacilli are gram-positive bacteria that are growing in importance for the healthcare industry and genetically engineering them as living therapeutics is highly sought after. However, progress in this field is hindered since most strains are difficult to genetically manipulate, partly due to their complex and thick cell walls limiting our capability to transform them with exogenous DNA. To overcome this, large amounts of DNA (>1 μg) are normally required to successfully transform these bacteria. An intermediate host, like E. coli, is often used to amplify recombinant DNA to such amounts although this approach poses unwanted drawbacks such as an increase in plasmid size, different methylation patterns and the limitation of introducing only genes compatible with the intermediate host. In this work, we have developed a direct cloning method based on in-vitro assembly and PCR amplification to yield recombinant DNA in significant quantities for successful transformation in L. plantarum WCFS1. The advantage of this method is demonstrated in terms of shorter experimental duration and the possibility to introduce a gene incompatible with E. coli into L. plantarum WCFS1