Biology subjects

Ashby, P. D.

Publications and source records attributed to Ashby, P. D..

4 recordsLinked to original sources

bioRxiv2026

SpyTag-Enabled Assembly of Bacterial Microcompartment Trimers into Macroscopic Layered Protein Materials

Protein self-assembly enables precise nanoscale organization but rarely translates into macroscopic materials that retain functionality beyond aqueous environments. Here, we report that a bacterial microcompartment (BMC) trimer fused with SpyTag (T1-SpyTag), when expressed as a standalone component, undergoes rapid and spontaneous self-assembly into macroscopically visible fibers and layered sheets. These structures span from the nanoscale to the millimeter scale, forming robust three-dimensional protein materials that remain structurally intact and functionally accessible in both solution and dried states. Unlike previously reported SpyTag-enabled BMC systems that function primarily as passive cargo-loading modules, T1-SpyTag macromolecular structures exhibit emergent material behavior, including chemical robustness under denaturing conditions, while preserving covalent reactivity toward SpyCatcher-fused cargos. The multilayered architecture enables tunable surface display, access to ultrathin, processable protein films, and surface renewability through layer-by-layer removal and regeneration. This work demonstrates how a minimal genetic modification of a native protein building block can drive the formation of functional, macroscopic protein materials, thus expanding the design space of BMC-derived assemblies for biointerfaces, catalysis, and sustainable protein materials engineering.

biochemistry↗

bioRxiv2022

Mechanical morphotype switching as an adaptive response in mycobacteria

Invading microbes face a myriad of cidal mechanisms of phagocytes that inflict physical damage to microbial structures. How intracellular bacterial pathogens adapt to these stresses is not fully understood. Here, we report a new virulence mechanism by which mycobacteria alter the mechanical stiffness of their cell surface to become refractory to killing during infection. Long-Term Time-Lapse Atomic Force Microscopy was used to reveal a process of "mechanical morphotype switching" in mycobacteria exposed to host intracellular stress. A "soft" mechanical morphotype switch enhances tolerance to intracellular macrophage stress, including cathelicidin. Genetic manipulation, by deletion of uvrA, or pharmacological treatment, with bedaquiline, locked mycobacteria into a "soft" mechanical morphotype state, enhancing survival in macrophages. Our study proposes microbial mechanical adaptation as a new axis for surviving host-mediated stressors. One-Sentence SummaryBacteria alter their cell surface mechanical properties to increase survival during macrophage infection.

microbiology↗

bioRxiv2021

A de novo matrix for macroscopic living materials from bacteria

Summary ParagraphEngineered living materials (ELMs) are composites of living cells embedded in a biopolymer matrix that combine the desirable properties of natural biomaterials with non-natural, tailored properties. ELMs with a wide range of sophisticated biological functions have been created by engineering the embedded cells using synthetic biology. Engineering a de novo biomolecular matrix would offer control over material assembly, structure, and composition, thus enabling us to grow macroscopic ELMs with customizable mechanical properties. However, we have lacked the genetic tools and design rules to genetically encode a synthetic matrix that programs collective cell self-organization into macroscopic structures. Here we report growth of macroscopic ELMs from Caulobacter crescentus cells that display and secrete an engineered self-interacting protein. This protein formed an extracellular de novo matrix and assembled cells into hierarchically-ordered, centimeter-scale ELMs. We showed that the mechanical, catalytic, and morphological properties of these ELMs can be tuned through genetic modification of the matrix. Our work identifies novel genetic tools, design and assembly rules for growing macroscopic ELMs with both wide-ranging mechanical properties and customizable functions. We anticipate the modularity of this approach will permit the incorporation of different protein polymers in the de novo matrix, thus allowing to generate ELMs with a variety of desired structures and compositions of the bulk material. We envision specific matrix properties that can be combined synergistically with existing cellular functions to greatly expand the opportunities for ELMs in human health, energy, and the environment.

synthetic biology↗

bioRxiv2020

Engineering high-yield biopolymer secretion creates an extracellular protein matrix for living materials

The bacterial extracellular matrix forms autonomously, giving rise to complex material properties and multicellular behaviors. Synthetic matrix analogues can replicate these functions, but require exogenously added material or have limited programmability. Here we design a two-strain bacterial system that self-synthesizes and structures a synthetic extracellular matrix of proteins. We engineered Caulobacter crescentus to secrete an extracellular matrix protein composed of elastin-like polypeptide (ELP) hydrogel fused to Supercharged SpyCatcher (SC(-)). This biopolymer was secreted at levels of 60 mg/L, an unprecedented level of biopolymer secretion by a gram-negative bacterium. The ELP domain was swapped with either a crosslinkable variant of ELP or resilin-like polypeptide, demonstrating this system is flexible. The SC(-)-ELP matrix protein bound specifically and covalently to the cell surface of a C. crescentus strain that displays a high-density array of SpyTag peptides via its engineered Surface-layer. Our work develops protein design rules for Type I secretion in C. crescentus, and demonstrates the autonomous secretion and assembly of programmable extracellular protein matrices, offering a path forward towards the formation of cohesive engineered living materials. IMPORTANCEEngineered living materials (ELM) aim to mimic characteristics of natural occurring systems, bringing the benefits of self-healing, synthesis, autonomous assembly, and responsiveness to traditional materials. Previous research has shown the potential of replicating the bacterial extracellular matrix (ECM) to mimic biofilms. However, these efforts require energy intensive processing or have limited tunability. We propose a bacterially-synthesized system that manipulates the protein content of the ECM, allowing for programmable interactions and autonomous material formation. To achieve this, we engineered a two-strain system to secrete a synthetic extracellular protein matrix (sEPM). This work is a step towards understanding the necessary parameters to engineering living cells to autonomously construct ELMs.

synthetic biology↗