Despite important breakthroughs in bottom‐up synthetic biology, a major challenge still remains the construction of free‐standing, macroscopic, and robust materials from protocell building blocks that are stable in water and capable of emergent behaviors. Herein, a new floating mold technique for the fabrication of millimeter‐ to centimeter‐sized protocellular materials (PCMs) of any shape that overcomes most of the current challenges in prototissue engineering is reported. Significantly, this technique also allows for the generation of 2D periodic arrays of PCMs that display an emergent non‐equilibrium spatiotemporal sensing behavior. These arrays are capable of collectively translating the information provided by the external environment and are encoded in the form of propagating reaction–diffusion fronts into a readable dynamic signal output. Overall, the methodology opens up a route to the fabrication of macroscopic and robust tissue‐like materials with emergent behaviors, providing a new paradigm of bottom‐up synthetic biology and biomimetic materials science.

The design and synthesis of a novel acid‐degradable polyethylene glycol‐based N‐hydroxysuccinimide (NHS) ester‐activated crosslinker is reported. The crosslinker is reactive towards nucleophiles and features a central ketal functional group that is stable at pH > 7.5 and rapidly hydrolyses at pH > 6.0. The crosslinker is used to (i) fabricate acid‐degradable polysaccharide hydrogels that exhibit controlled degradation upon exposure to an acidic environment or via endogenous enzyme activity; and (ii) construct hydrogel‐filled protein‐polymer microcompartments (termed proteinosomes) capable of pH‐dependent membrane disassembly. Taken together the results provide new opportunities for the fabrication of pH‐responsive soft materials with potential applications in drug delivery, tissue engineering, and soft‐matter bioengineering.

Silk fibroin (SF) is one of the most exploited and studied natural materials, yet the complex sequence of dynamic transitions required to transform from a liquid state to a solid fibre remain unresolved. We found that fibroin heavy chain (FibH) from Bombyx mori (B. mori), a multidomain protein with highly repetitive domains, folds to form β-solenoid structures. The single fibroin molecule undergoes conformational transformations from a globular form to an extended solenoid driven by pH reduction. Following this, the N-terminal domain (NTD) drives higher-order oligomerisation beyond the proposed biological tetrameric unit when in the extended state. The proposed supramolecular structures explain the complex rheological behaviour observed for silk and the liquid crystalline textures observed within the B. mori gland. Overall, our results demonstrate that silk assembly is a highly organised process, where a mild reduction in pH promotes molecular reorganisation and higher-order oligomerisation of the N-terminal domain into structures that precede fibre formation. Moreover, we suggest that NTD is at least partially hydrolysed and lost during the standard regeneration/reconstitution processing, explaining the historical differences between native and regenerated feedstocks. We believe our results expand our understanding of the liquid-to-solid transformation of fibroin molecules, as well as indicate to other researchers a clear strategy to determine the quality of their feedstock, which can be translated into improved materials fabrication.

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