A de novo matrix for macroscopic living materials from bacteria

Engineered living materials (ELMs) embed living cells in a biopolymer matrix to create novel materials with tailored functions. While bottom-up assembly of macroscopic ELMs with a de novo matrix would offer the greatest control over material properties, we lack the ability to genetically encode a protein matrix that leads to collective self-organization. Here we report growth of ELMs from Caulobacter crescentus cells that display and secrete a self-interacting protein. This protein formed a de novo matrix and assembled cells into centimeter-scale ELMs. Discovery of design and assembly principles allowed us to tune the mechanical, catalytic, and morphological properties of these ELMs. This work provides novel tools, design and assembly rules, and a platform for growing ELMs with control over matrix and cellular structure and function.

Unsettled Technology Areas in Deterministic Assembly Approaches for Industry 4.0

Increased production rates and cost reduction are affecting manufacturing in all sectors of the mobility industry. One enabling methodology that could achieve these goals in the burgeoning “Industry 4.0” environment is the deterministic assembly (DA) approach. The DA approach is defined as an optimized assembly process; it always forms the same final structure and has a strong link to design-for-assembly and design-for-automation methodologies. It also looks at the whole supply chain, enabling drastic savings at the original equipment manufacturer (OEM) level by reducing recurring costs and lead time. Within Industry 4.0, DA will be required mainly for the aerospace and the space industry, but serves as an interesting approach for other industries assembling large and/or complex components. In its entirety, the DA approach connects an entire supply chain—from part manufacturing at an elementary level to an OEM’s final assembly line level. Addressing the whole process of aircraft design and manufacturing is necessary to develop further collaboration models between OEMs and the supply chain, including addressing the most pressing technology challenges. Since all parts aggregate at the OEM level, the OEM—as an integrator of all these single parts—needs special end-to-end methodologies to drastically decrease cost and lead time. This holistic approach can be considered in part design as well (in the design-for-automation and design-for-assembly philosophy). This allows for quicker assembly at the OEM level, such as “part-to-part” or “hole-to-hole” approaches, versus traditional, classical assembly methods like manual measurement or measurement-assisted assembly. In addition, it can increase flexibility regarding rate changes in production (such as those due to pandemic- or climate-related environmental challenges). The standardization and harmonization of these areas would help all industries and designers to have a deterministic approach with an end-to-end concept. Simulations can easily compare possible production and assembly steps with different impacts on local and global tolerances. Global measurement feedback needs high-accuracy turnkey solutions, which are very costly and inflexible. The goal of standardization would be to use Industry 4.0 feedback and features, as well as to define several building blocks of the DA approach as a one-way assembly (also known as one-up assembly, or “OUA”), false one-way assembly, “Jig-as-Master,” etc., up to the hole-to-hole assembly approach. The evolution of these assembly principles and the link to simulation approaches are undefined and unsolved domains; they are discussed in this report. They must be discussed in greater depth with aims of (first) clarifying the scope of the industry-wide alignment needs and (second) prioritizing the issues requiring standardization. NOTE: SAE EDGE™ Research Reports are intended to identify and illuminate key issues in emerging, but still unsettled, technologies of interest to the mobility industry. The goal of SAE EDGE™ Research Reports is to stimulate discussion and work in the hope of promoting and speeding resolution of identified issues. SAE EDGE™ Research Reports are not intended to resolve the challenges they identify or close any topic to further scrutiny.

Assembly principles and stoichiometry of a complete human kinetochore module

Centromeres are epigenetically determined chromosomal loci that seed kinetochore assembly to promote chromosome segregation during cell division. CENP-A, a centromere-specific histone H3 variant, establishes the foundations for centromere epigenetic memory and kinetochore assembly. It recruits the constitutive centromere-associated network (CCAN), which in turn assembles the microtubule-binding interface. How the specific organization of centromeric chromatin relates to kinetochore assembly and to centromere identity through cell division remains conjectural. Here, we break new ground by reconstituting a functional full-length version of CENP-C, the largest human CCAN subunit and a blueprint of kinetochore assembly. We show that full-length CENP-C, a dimer, binds stably to two nucleosomes and permits further assembly of all other kinetochore subunits in vitro with relative ratios closely matching those of endogenous human kinetochores. Our results imply that human kinetochores emerge from clustering multiple copies of a fundamental module and may have important implications for transgenerational inheritance of centromeric chromatin.

Characterizing the Structure and Interactions of Model Lipid Membranes Using Electrophysiology

The cell membrane is a protective barrier whose configuration determines the exchange both between intracellular and extracellular regions and within the cell itself. Consequently, characterizing membrane properties and interactions is essential for advancements in topics such as limiting nanoparticle cytotoxicity. Characterization is often accomplished by recreating model membranes that approximate the structure of cellular membranes in a controlled environment, formed using self-assembly principles. The selected method for membrane creation influences the properties of the membrane assembly, including their response to electric fields used for characterizing transmembrane exchanges. When these self-assembled model membranes are combined with electrophysiology, it is possible to exploit their non-physiological mechanics to enable additional measurements of membrane interactions and phenomena. This review describes several common model membranes including liposomes, pore-spanning membranes, solid supported membranes, and emulsion-based membranes, emphasizing their varying structure due to the selected mode of production. Next, electrophysiology techniques that exploit these structures are discussed, including conductance measurements, electrowetting and electrocompression analysis, and electroimpedance spectroscopy. The focus of this review is linking each membrane assembly technique to the properties of the resulting membrane, discussing how these properties enable alternative electrophysiological approaches to measuring membrane characteristics and interactions.

4 SPECIES OF BACTERIA DETERMINISTICALLY FORM A STABLE BIOFILM IN A MILLIFLUIDIC CHANNEL: ASSEMBLY PRINCIPLES

Multispecies microbial adherent communities are widespread in nature and organisms but the principles of their assembly and development remain unclear. Yet, the demand to understand and predict the responses of such living communities to environmental changes is increasing, calling for new approaches. Here, we test the possibility to establish a simplified but relevant model of multispecies biofilm in a laboratory setup enabling in situ real-time monitoring of the community development and control of the environmental parameters in order to decipher the mechanisms underlying the formation of the community. Using video-microscopy and species combinatorial approach, we assess the global and individual species spatiotemporal development in millifluidic channels under constant flow of nutrients. Based on quantitative measurements of expansion kinetics, local dynamics and spatial distribution, we demonstrate that the four chosen species (Bacillus thuringiensis, Pseudomonas fluorescens, Kocuria varians and Rhodocyclus sp.) form a dynamical community that deterministically reaches its equilibrium after about 30 hours of growth. We evidence the emergence of complexity in this simplified community as reported by spatial heterogeneity rise and non-monotonic developmental kinetics. We find interspecies interactions consisting in competition for resources - in particular oxygen - and both direct and indirect physical interactions but no positive feedback. Thereby, we introduce a model of multispecies adherent community where effective couplings result from individual species quest for fitness optimization in a moving and heterogenous environment. This control and the understanding of this simplified experimental model shall open new avenues to apprehend adherent bacterial communities behavior in a context of rapid global change.

Virus-Like Particles Produced Using the Brome Mosaic Virus Recombinant Capsid Protein Expressed in a Bacterial System

Virus-like particles (VLPs), due to their nanoscale dimensions, presence of interior cavities, self-organization abilities and responsiveness to environmental changes, are of interest in the field of nanotechnology. Nevertheless, comprehensive knowledge of VLP self-assembly principles is incomplete. VLP formation is governed by two types of interactions: protein–cargo and protein–protein. These interactions can be modulated by the physicochemical properties of the surroundings. Here, we used brome mosaic virus (BMV) capsid protein produced in an E. coli expression system to study the impact of ionic strength, pH and encapsulated cargo on the assembly of VLPs and their features. We showed that empty VLP assembly strongly depends on pH whereas ionic strength of the buffer plays secondary but significant role. Comparison of VLPs containing tRNA and polystyrene sulfonic acid (PSS) revealed that the structured tRNA profoundly increases VLPs stability. We also designed and produced mutated BMV capsid proteins that formed VLPs showing altered diameters and stability compared to VLPs composed of unmodified proteins. We also observed that VLPs containing unstructured polyelectrolyte (PSS) adopt compact but not necessarily more stable structures. Thus, our methodology of VLP production allows for obtaining different VLP variants and their adjustment to the incorporated cargo.

Collagen Pentablock Copolymers Form Smectic Liquid Crystals as Precursors for Mussel Byssus Fabrication

Protein-based biological materials are important role models for the design and fabrication of next generation advanced polymers. Marine mussels (Mytilus spp.) fabricate hierarchically structured collagenous fibers known as byssal threads via bottom-up supramolecular assembly of fluid protein precursors. The high degree of structural organization in byssal threads is intimately linked to their exceptional toughness and self-healing capacity. Here, we investigated the hypothesis that multidomain collagen precursor proteins, known as preCols, are stored in secretory vesicles as a colloidal liquid crystal (LC) phase prior to thread self-assembly. Using advanced electron microscopy methods, including scanning TEM and FIB-SEM, we visualized the detailed smectic preCol LC nanostructure in 3D, including various LC defects, confirming this hypothesis and providing quantitative insights into the mesophase structure. In light of these findings, we performed an in-depth comparative analysis of preCol protein sequences from multiple Mytilid species revealing that the smectic organization arises from an evolutionarily conserved ABCBA penta-block co-polymer-like primary structure based on demarcations in hydropathy and charge distribution, as well as terminal pH-responsive domains<br>that trigger fiber formation. These distilled supramolecular assembly principles provide inspiration and strategies for sustainable assembly of nanostructured polymeric materials for<br>potential applications in engineering and biomedical applications.

Collagen Pentablock Copolymers Form Smectic Liquid Crystals as Precursors for Mussel Byssus Fabrication

Protein-based biological materials are important role models for the design and fabrication of next generation advanced polymers. Marine mussels (Mytilus spp.) fabricate hierarchically structured collagenous fibers known as byssal threads via bottom-up supramolecular assembly of fluid protein precursors. The high degree of structural organization in byssal threads is intimately linked to their exceptional toughness and self-healing capacity. Here, we investigated the hypothesis that multidomain collagen precursor proteins, known as preCols, are stored in secretory vesicles as a colloidal liquid crystal (LC) phase prior to thread self-assembly. Using advanced electron microscopy methods, including scanning TEM and FIB-SEM, we visualized the detailed smectic preCol LC nanostructure in 3D, including various LC defects, confirming this hypothesis and providing quantitative insights into the mesophase structure. In light of these findings, we performed an in-depth comparative analysis of preCol protein sequences from multiple Mytilid species revealing that the smectic organization arises from an evolutionarily conserved ABCBA penta-block co-polymer-like primary structure based on demarcations in hydropathy and charge distribution, as well as terminal pH-responsive domains<br>that trigger fiber formation. These distilled supramolecular assembly principles provide inspiration and strategies for sustainable assembly of nanostructured polymeric materials for<br>potential applications in engineering and biomedical applications.

Assembly principles and stoichiometry of a complete human kinetochore module

Centromeres are epigenetically determined chromosomal loci that seed kinetochore assembly to promote chromosome segregation during cell division. CENP-A, a centromere-specific histone H3 variant, establishes the foundations for centromere epigenetic memory and kinetochore assembly. It recruits the constitutive centromere-associated network (CCAN), which in turn assembles the microtubule-binding interface. How the specific organization of centromeric chromatin relates to kinetochore assembly and to centromere identity through cell division remains conjectural. Here, we break new ground by reconstituting a functional full-length version of CENP-C, the largest human CCAN subunit and a blueprint of kinetochore assembly. We show that full-length CENP-C, a dimer, binds stably to two nucleosomes, and permits further assembly of all other kinetochore subunits in vitro with relative ratios that closely match those of endogenous human kinetochores. Our results imply that human kinetochores emerge from clustering multiple copies of a fundamental module, and may have important implications for trans-generational inheritance of centromeric chromatin.