ACS Synth. Biol. 8, 181–190 (2019)

Surface-layer (S-layer) proteins form regular lattices on the external surfaces of many cells. In the bacterium Caulobacter crescentus, the S-layer protein RsaA forms a hexameric lattice, and previous work has demonstrated that it tolerates insertions of peptides and protein domains. Charrier et al. now demonstrate the use of RsaA as a platform for engineering living biomaterials by employing the SpyTag–SpyCatcher split protein system. Following expression of an RsaA variant with an inserted SpyTag sequence, incubation with a SpyCatcher-tagged protein results in the covalent, irreversible attachment of desired functionalities to the lattice. The SpyTag sequence can be inserted at any of eight positions in RsaA without disrupting the expression and patterning of the S-layer protein; however, this positioning affects the density of SpyCatcher modification, providing versatility in the biomaterial structure. The authors used this system to build ordered biomaterials on the C. crescentus surface with elastin-like polypeptide or quantum dots without compromising cellular viability. This approach could potentially be adapted for engineering S-layer proteins in other organisms and be developed further to produce increasingly complex biomaterials.

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