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ABSTRACT Novel approaches are needed to study relationships between oral biofilm strains, enable three-dimensional oral biofilm deposition, and hasten the rigor and pace of basic and translational biofilm studies. Previously, 3D-bioprinters were leveraged to deposit spatially patterned biofilms onto sugar-rich agar surfaces to study how the underlying spatial organization of various microbes impacts biofilm persistence and virulence. Herein, we have developed a new method to adapt this process from limited, soft agar surfaces to biomimetic solid substrates submerged in aqueous solutions for studying oral biofilmsin vitro. Streptococcus mutansUA159 was used to compare standardin vitrobiofilm development with our new 3D-printed bio-ink hydrogels on hydroxyapatite disks, which mimic tooth surfaces. Biofilms formed using the bio-ink methodology showed minimal quantitative differences in virulence factors, including environmental pH, biomass, and cell density, compared to biofilms formed using the standardin vitromethodology. The bio-ink technique resulted in higher exopolysaccharide deposition, a key virulence factor for biofilm cohesion and protection, as well as more homogeneous spatial distribution of bacterial microcolonies. Our newly developed technique produces 3D-printable model biofilms that match the virulence benchmarks of the standard method, opening possibilities to print biofilms onto any substrate and a new way to study multidimensional biofilm dynamics.IMPORTANCEDental caries is the most common oral disease caused by biofilms in humans with cost limitations. Changes in the human diet have increased the exposure to sugar-rich processed food, increasing the incidence and severity of dental caries and creating greater rationale for understanding biofilm deposition, microbial interactions, and maintenance of quiescence of the oral microbiota. Recent 3D-printing techniques have been leveraged to develop the first model biofilms, providing spatial control over microbe deposition and enabling unprecedented investigation of the impact of cell-cell interactions and spatial organizationupon biofilm persistence, sensitivity to drugs, and virulence. Here, we have developed new methods to extend bioprinting to oral biofilms using cariogenicStreptococcus mutans. Our technique is an attempt to establish an alternative method for oral biofilm formationin vitrothat uses 3D-printing tools, preserving the virulence of standardin vitrobiofilms while amplifying the availability and versatility of methods for understanding the microbiome. 

ABSTRACT The draft genome of a chemolithoautotrophic ammonia-oxidizing bacterium of the genusNitrosomonasis reported.Nitrosomonassp. strain ANs5, previously classified as a strain ofN. halophila, is an alkali-tolerant ammonia-oxidizing bacterium isolated from the soda lakes of northeast Mongolia. 

Abstract Recently, engineered bacterial cells have been shown to behave as optically-active photonic devices comparable to industrially fabricated microlenses¹. Bacterial cells can be encapsulated within a layer of polysilicate through surface display of the sea sponge enzyme silicatein, which mineralizes a polysilicate coating. The addition of this polysilicate layer significantly enhances the ability of these cells to guide, scatter, and focus light¹. However, this previous technique was limited to creating rod-shaped microlenses, which are not ideal for all applications. Here we expand upon this technology by engineering the shapes of silicatein-displaying bacterial cells. Through the overexpression of the genesbolA^2–5andsulA^6,7or through the use of the drug A22^8,9, we are able to alterEscherichia colicells from their characteristic rod-like shape to either spherical or filamentous forms. Round cells encapsulated in polysilicate were shown to scatter light more intensely and symmetrically than rod-shaped cells, while encapsulated filamentous cells were shown to guide light similarly to an optical fiber. This control over the size and shape of optically-active cells is a major advancement towards developing bio-engineered photonic devices such as nanophotonic waveguides, spherical microlens arrays, and advanced biosensors. 

Abstract Three dimensional printing has emerged as a widely acceptable strategy for the fabrication of mammalian cell laden constructs with complex microenvironments for tissue engineering and regenerative medicine. More recently 3D printed living materials containing microorganisms have been developed and matured into living biofilms. The potential for engineered 3D biofilms as in vitro models for biomedical applications, such as antimicrobial susceptibility testing, and environmental applications, such as bioleaching, bioremediation, and wastewater purification, is extensive but the need for an in‐depth understanding of the structure–function relationship between the complex construct and the microorganism response still exists. This review discusses 3D printing fabrication methods for engineered biofilms with specific structural features. Next, it highlights the importance of bioink compositions and 3D bioarchitecture design. Finally, a brief overview of current and potential applications of 3D printed biofilms in environmental and biomedical fields is discussed. 

The severe, long-lasting harm caused by plastic pollution to marine ecosystems and coastal economies has led to the development of biodegradable plastics; however, their limited decomposition in cold, dark marine environments remains a challenge. Here, we present our newly developed technologies for creating 3D-bioprinted living materials for bioplastic degradation with specific use in marine environments. Our approach integrates halotolerant bioplastic-degrading bacteriumBacillussp. NRRL B-14911 into alginate-based bio-ink to print an engineered living material (ELM) termed a “bio-sticker.” Quantification of bacteria viability reveals that bioprinted marine bacteria survive within bio-stickers for more than three weeks. The rate at which the bio-stickers degrade the bioplastic polyhydroxybutyrate (PHB) can be tuned by altering bio-sticker biomass concentration, bioplastic concentration, or incubation temperature. Bio-stickers that are transferred to a new PHB sample still retain high biodegradation activity, demonstrating their durability. Strain sweep oscillatory tests demonstrate viscoelastic behavior of the bio-stickers. Monotonic tensile tests indicate that the elastic modulus and the adhesion of the bio-stickers are not negatively impacted by bacteria growth or incubation temperature. Our work paves the way for development of ELMs to facilitate the inclusion of bioplastics within the blue economy, promoting the emergence of more sustainable and eco-friendly materials. 

Cutting-edge photonic devices frequently rely on microparticle components to focus and manipulate light. Conventional methods used to produce these microparticle components frequently offer limited control of their structural properties or require low-throughput nanofabrication of more complex structures. Here, we employ a synthetic biology approach to produce environmentally friendly, living microlenses with tunable structural properties. We engineeredEscherichia colibacteria to display the silica biomineralization enzyme silicatein from aquatic sea sponges. Our silicatein-expressing bacteria can self-assemble a shell of polysilicate “bioglass” around themselves. Remarkably, the polysilicate-encapsulated bacteria can focus light into intense nanojets that are nearly an order of magnitude brighter than unmodified bacteria. Polysilicate-encapsulated bacteria are metabolically active for up to 4 mo, potentially allowing them to sense and respond to stimuli over time. Our data demonstrate that synthetic biology offers a pathway for producing inexpensive and durable photonic components that exhibit unique optical properties.