Person: Tay, Pei Kun Richie Richie
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Tay
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Pei Kun Richie Richie
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Tay, Pei Kun Richie Richie
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Publication Scalable Production of Genetically Engineered Nanofibrous Macroscopic Materials via Filtration(American Chemical Society (ACS), 2016-10-26) Dorval Courchesne, Noemie-Manuelle; Duraj-Thatte, Anna; Tay, Pei Kun Richie Richie; Nguyen, Peter; Joshi, NeelAs interest in using proteins to assemble functional, biocompatible and environmentally- friendly materials is growing, developing scalable protocols for producing recombinant proteins coupled to straightforward fabrication processes is becoming crucial. Here, we use E. coli bacteria to produce amyloid protein nanofibers that are key constituents of the biofilm extracellular matrix, and show that protein nanofiber aggregates can be purified using a fast and easily accessible vacuum filtration procedure. With their high resistance to heat, detergents, solvents and denaturing agents, engineered curli nanofibers remain functional throughout the rigorous processing, and can be used to assemble macroscopic materials. As a demonstration, we show that engineered curli nanofibers can be fabricated into self-standing films while maintaining the functionality of various fused domains that confer new specific binding activity to the material. We also demonstrate that purified curli fibers can be disassembled, reassembled into thin films, and recycled for further materials processing. We envision this scheme as an easily adoptable method for those interested in the scalable production of engineered protein- based materials.Publication Programmable biofilm-based materials from engineered curli nanofibres(Nature Publishing Group, 2014) Nguyen, Peter; Botyanszki, Zsofia; Tay, Pei Kun Richie Richie; Joshi, NeelThe significant role of biofilms in pathogenicity has spurred research into preventing their formation and promoting their disruption, resulting in overlooked opportunities to develop biofilms as a synthetic biological platform for self-assembling functional materials. Here we present Biofilm-Integrated Nanofiber Display (BIND) as a strategy for the molecular programming of the bacterial extracellular matrix material by genetically appending peptide domains to the amyloid protein CsgA, the dominant proteinaceous component in Escherichia coli biofilms. These engineered CsgA fusion proteins are successfully secreted and extracellularly self-assemble into amyloid nanofibre networks that retain the functions of the displayed peptide domains. We show the use of BIND to confer diverse artificial functions to the biofilm matrix, such as nanoparticle biotemplating, substrate adhesion, covalent immobilization of proteins or a combination thereof. BIND is a versatile nanobiotechnological platform for developing robust materials with programmable functions, demonstrating the potential of utilizing biofilms as large-scale designable biomaterials.Publication Engineered catalytic biofilms: Site-specific enzyme immobilization onto E. coli curli nanofibers(Wiley-Blackwell, 2015) Botyanszki, Zsofia; Tay, Pei Kun Richie Richie; Nguyen, Peter; Nussbaumer, Martin; Joshi, NeelBiocatalytic transformations generally rely on purified enzymes or whole cells to perform complex transformations that are used on industrial scale for chemical, drug, and biofuel synthesis, pesticide decontamination, and water purification. However, both of these systems have inherent disadvantages related to the costs associated with enzyme purification, the long-term stability of immobilized enzymes, catalyst recovery, and compatibility with harsh reaction conditions. We developed a novel strategy for producing rationally designed biocatalytic surfaces based on Biofilm Integrated Nanofiber Display (BIND), which exploits the curli system of E. coli to create a functional nanofiber network capable of covalent immobilization of enzymes. This approach is attractive because it is scalable, represents a modular strategy for site-specific enzyme immobilization, and has the potential to stabilize enzymes under denaturing environmental conditions. We site-specifically immobilized a recombinant α-amylase, fused to the SpyCatcher attachment domain, onto E. coli curli fibers displaying complementary SpyTag capture domains. We characterized the effectiveness of this immobilization technique on the biofilms and tested the stability of immobilized α-amylase in unfavorable conditions. This enzyme-modified biofilm maintained its activity when exposed to a wide range of pH and organic solvent conditions. In contrast to other biofilm-based catalysts, which rely on high cellular metabolism, the modified curli-based biofilm remained active even after cell death due to organic solvent exposure. This work lays the foundation for a new and versatile method of using the extracellular polymeric matrix of E. coli for creating novel biocatalytic surfaces. Biotechnol. Bioeng. 2015;112: 2016–2024. © 2015 Wiley Periodicals, Inc.Publication A Synthetic Circuit for Mercury Bioremediation Using Self-Assembling Functional Amyloids(American Chemical Society (ACS), 2017) Tay, Pei Kun Richie Richie; Nguyen, Peter; Joshi, NeelSynthetic biology approaches to bioremediation are a key sustainable strategy to leverage the self-replicating and programmable aspects of biology for environmental stewardship. The increasing spread of anthropogenic mercury pollution into our habitats and food chains is a pressing concern. Here, we explore the use of programmed bacterial biofilms to aid in the sequestration of mercury. We demonstrate that by integrating a mercury-responsive promoter and an operon encoding a mercury-absorbing self-assembling extracellular protein nanofiber, we can engineer bacteria that can detect and sequester toxic Hg2+ ions from the environment. This work paves the way for the development of on-demand biofilm living materials that can operate autonomously as heavy-metal absorbents.