(D-121) Production of functional living hydrogel with amyloid assemblies

In recent years, the development of synthetic biology has introduced the concept of coupling engineered living cells with matrix materials to create sophisticated and versatile living materials[1][2] with programmable features. Hydrogel, a crosslinked material[3][4] widely used in biomedical area due to its remarkable properties including biocompatibility and mechanical strength, is one of the commonly used living material matrices. Amyloids[5][6], once known as disease-related proteins, are now recognized for their unique mechanical characteristics and important functional roles in various living organisms. Their genetic engineering potential makes them an excellent platform for creating engineered proteins with desired functions, enabling them to combine with other materials to create living materials with enhanced properties and diverse functions. Here, we propose to utilize bacteria encapsulated in chitosan hydrogel to produce a living hydrogel material with secondary network of amyloids distributed throughout, which could be responsive to external signals such as temperature and pH. In the fabrication process, a chitosan network was first constructed to serve as the initial network, capturing engineered bacteria containing the ePop circuit[7], which causes cell lysis at a specific cell density and releases the expressed functional amyloid proteins inside the cells. Then the amyloids self-assembled within the chitosan network to create the double network hydrogel. This living hydrogel can acquire enhanced mechanical property with the amyloid network and has a self-repairing ability with the living cells auto-lysis system. Various functional proteins can be easily introduced into the system through genetic engineering by tagging the proteins of interest with amyloids.

To create the chitosan-amyloid double network living hydrogel. we followed a previously reported electrohydrodynamic spraying-like method [8] to generate chitosan-based hydrogel microparticles. Escherichia coli (E. coli) programmed to auto-lysis and release amyloids and functional proteins were encapsulated inside the chitosan capsules during the fabrication process. The capsules were then incubated overnight to allow cell growth and protein expression, leading to the formation of an inner self-assembled functional amyloid network. The amyloids used in the study are yeast stress regulation proteins-Cdc19 [9][10], known for their ability to form self-assembled structures and respond to changes in pH. Two Cdc19 proteins were fused through elastin-like polypeptides to serve as the basic unit of the amyloid network. In current study, mCherry proteins and beta-lactamase were selected as functional proteins, tagged by the Cdc19 and introduced into the system by coculturing all the engineered strains within the hydrogel. ThioflavinT (ThT) staining was used to observe the formation and pH responsiveness of the amyloid network structure, while fluorescence microscopy experiments and beta-lactamase enzyme activity tests were conducted to evaluate the material's protection ability and protein anchoring capability.

ThT staining results demonstrated that the engineered Cdc19 proteins and Cdc19-tagged functional proteins, released from the encapsulated cells can be triggered to form micron-scale network structures in low pH environments, which indicates that Cdc19 proteins hold promise for creating amyloid network within the chitosan hydrogel, with the potential for pH-responsive control. Fluorescence microscopy experiments further confirmed the hydrogel's ability to capture Cdc19-tagged proteins, such as anchoring fluorescence proteins inside the capsules. Chitosan-amyloid capsules had significantly reduced fluorescence out of the capsules, approximately one-third of the non-amyloid chitosan capsules after overnight culture, indicating effective protein anchoring. Moreover, preliminary results suggested low pH treatment at different step during the fabrication process may influence the protein anchoring ability of the capsules. We have successfully produced the chitosan-amyloid hydrogel capsules which can effectively encapsulate functional proteins and display responsiveness to varying pH conditions. In the next step, enzyme activity tests with chitosan-amyloid capsules containing beta-lactamase will be conducted to evaluate capsules' protective properties. Additionally, further examination of the pH-responsive behavior of the amyloid structure inside the hydrogel is essential. While the material exhibited some pH responsiveness, it behaved differently from pure Cdc19 protein, notably in the irreversibility of the low pH-triggered structures. More research is needed to determine if precise control of the structure formation can be achieved through different pH conditions. Overall, we believe this chitosan-amyloid living hydrogel represents a meaningful addition to the field of living materials. The genetically modified Cdc19 protein can help enhance the material structure and introduce functional proteins into the hydrogel system, while the auto-lysis system enhances the self-healing properties of the material and provides a broader option of accessible proteins by avoiding the transmembrane transport issues.

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[10] Cereghetti, G. et al. A conserved mechanism regulates reversible amyloids via pH-sensing regions. bioRxiv 2022.03.21.484600 (2022).