Fluorescence Microscopy

Encapsulation of microbes in natural or synthetic matrices is a key aspect of engineered living materials, although the influence of such confinement on microbial behavior is poorly understood. A few recent studies have shown that the spatial confinement and mechanical properties of the encapsulating material significantly influence microbial behavior, including growth, metabolism, and gene expression. However, comparative studies within different bacterial species under identical confinement conditions are limited. In this study, Gram-negative Escherichia coli Nissle 1917 and Gram-positive Lactiplantibacillus plantarum WCFS1 were encapsulated in hydrogel matrices, and their growth, metabolic activity, and recombinant gene expression were examined under varying degrees of hydrogel stiffness, achieved by adjusting the polymer concentration and chemical cross-linking. Both bacteria grow from single cells into confined colonies, but more interestingly, in E. coli gels, mechanical properties influenced colony growth, size, and morphology, whereas this did not occur in L. plantarum gels. However, with both bacteria, increased matrix stiffness led to higher levels of recombinant protein production within the colonies. By measuring metabolic heat from the bacterial gels using the isothermal microcalorimetry technique, it was inferred that E. coli adapts to the mechanical restrictions through multiple metabolic transitions and is significantly affected by the different hydrogel properties. Contrastingly, both of these aspects were not observed with L. plantarum. These results revealed that despite both bacteria being gut-adapted probiotics with similar geometries, mechanical confinement affects them considerably differently. The weaker influence of matrix stiffness on L. plantarum is attributed to its slower growth and thicker cell wall, possibly enabling the generation of higher turgor pressures to overcome restrictive forces under confinement. By providing fundamental insights into the interplay between mechanical forces and bacterial physiology, this work advances our understanding of how matrix properties shape bacterial behavior. The implications of these findings will aid the design of engineered living materials for therapeutic applications.

Living microbial therapeutics promise precise, programmable interventions at disease sites, yet most demonstrations of on demand drug release still rely on Escherichia coli, whose rich genetic toolkit is unmatched among probiotics. In particular, genetic parts to regulate in situ protein production are severely lacking in non-model probiotic bacteria like lactobacilli. Here, we equip the probiotic Lactiplantibacillus plantarum with high-performance genetic switches and show how material encapsulation can further enhance their behavior. By integrating cumate or vanillate-responsive operators and repressors with the strongest constitutive promoter in L. plantarum (Ptec), we generated two switches that support micromolar range induction. In rapidly growing culture conditions, acidification-associated leakiness of the switch was observed, which could compromise their applicability for precise on-demand delivery of drugs. Furthermore, such leakiness also limits the duration for which these engineered probiotics can be reliably used. By restricting growth through mild temperature or nutrient limitation, acidification and leakiness were suppressed. Strikingly, immobilizing the engineered cells in core-shell alginate beads (Protein Eluting Alginate with Recombinant Lactobacilli, PEARLs) almost eliminated leakiness, enabling day-scale, reversible control of intracellular reporters and secreted enzymes. This leakiness suppression persisted when two strains carrying orthogonal switches were co-encapsulated and even after miniaturization to submillimeter beads. These results expand the genetic toolbox of probiotic L. plantarum, demonstrate the synergy between genetic circuit design and material encapsulation, and advance lactobacilli toward stimuli-responsive therapeutic platforms.

The 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.

Microbial biofactories allow the upscaled production of high-value compounds in biotechnological processes. This is particularly advantageous for compounds like flavonoids that promote better health through their antioxidant, antibacterial, anticancer and other beneficial effects but are only produced in small quantities in their natural plant-based hosts. Bacteria like E. coli have been genetically modified with enzyme cascades to produce flavonoids like naringenin and pinocembrin from coumaric or cinnamic acid. Despite advancements in yield optimization, the production of these compounds still involves high costs associated with their biosynthesis, purification, storage and transport. An alternative production strategy could involve the direct delivery of the microbial biofactories to the body. In such a strategy, ensuring biocontainment of the engineered microbes in the body and controlling production rates are major challenges. In this study, these two aspects are addressed by developing engineered living materials (ELMs) consisting of probiotic microbial biofactories encapsulated in biocompatible hydrogels. Engineered probiotic E. coli Nissle 1917 able to efficiently convert cinnamic acid into pinocembrin were encapsulated in poly(vinyl alcohol)-based hydrogels. The biofactories are contained in the hydrogels for a month and remain metabolically active during this time. Control over production levels is achieved by the containment inside the material, which regulates bacteria growth, and by the amount of cinnamic acid in the medium.

Adherens junctions (AJs) allow cell contact to inhibit epithelial migration yet also permit epithelia to move as coherent sheets. How, then, do cells identify which contacts will inhibit locomotion? Here, we show that in human epithelial cells this arises from the orientation of cortical flows at AJs. When the leader cells from different migrating sheets make head-on contact with one another, they assemble AJs that couple together oppositely directed cortical flows. This applies a tensile signal to the actin-binding domain (ABD) of α-catenin, which provides a clutch to promote lateral adhesion growth and inhibit the lamellipodial activity necessary for migration. In contrast, AJs found between leader cells in the same migrating sheet have cortical flows aligned in the same direction, and no such mechanical inhibition takes place. Therefore, α-catenin mechanosensitivity in the clutch between E-cadherin and cortical F-actin allows cells to interpret the direction of motion via cortical flows and signal for contact to inhibit locomotion.