For small-scale spinning of composite silk fusion protein and CNF fibers, an in-house custom-made experimental setup (fig. S2) consisting of five main parts was used: a pair of pumps, a pair of sample containers, a micro static mixer assembly, capillary tubing, and a coagulation bath (96% ethanol). For larger-scale spinning, a motorized take up spinning system consisting of AC motors, pulleys, drying fans, and collecting rollers was included downstream. Before use, the CNF and recombinantly produced protein solutions were loaded in each sample container, centrifuged for 10 min at 2000 rpm (720g) at 25°C to avoid defects caused by air bubbles, and then connected to each pump. Using the high-performance pumps with fine adjustment of flow rate (volumetric flow rate, 500 μl/min; linear flow rate, 150 cm/min), eluent was separately delivered to each sample container, building a pressure behind movable seals and forcing the CNF and protein solutions through separate capillary tubing with internal diameters of 500 μm. The outlet of the mixing tee was connected to capillary tubing with the internal diameter of 500 μm and the length of 150 cm as described before (16). The protein-CNF solution was then pushed through the capillary tubing into a coagulation bath of ethanol to precipitate out and form solid fibers.

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