(178a) Efficient Assembly of Genes That Encode Repetitive Fusion Protein Biomaterials

Nature provides a rich source of functional polymers, which display properties often unattainable with synthetic polymers. In particular, repetitive proteins exhibit diverse and highly tunable behavior owing to their sequence-defined nature. The precise control over protein sequence makes repetitive protein-based materials promising candidates for engineering stimuli-responsive soft matter. A challenge to synthesizing repetitive proteins is assembling the repetitive genes encoding for them; inefficiencies in repetitive gene assembly impede rapid iteration through material synthesis and characterization to achieve materials with desired properties. Herein, we report a nested strategy for gene assembly of a repetitive fusion protein to efficiently assemble a panel of repetitive fusion protein variants.

Genes encoding repetitive fusion proteins were assembled using sequential Golden Gate assembly reactions. We sought to generate a protein with two unique repetitive blocks separated by a variable linker region. Both distinct blocks were assembled from gene fragments using Golden Gate assembly. The region between the two blocks contained recognition sites for a second Golden Gate enzyme, enabling the rapid insertion of modular linker sequences via a single fragment assembly. Single fragment assemblies typically display higher fidelity than multi-fragment assemblies; this nested assembly approach allows the generation of multiple variants with unique linker sequences using only a single round of multi-fragment assembly for the repetitive blocks. In conclusion, we demonstrate a nested gene assembly approach that exploits two Golden Gate enzymes to rapidly synthesize variants of repetitive fusion protein biomaterials.