Directed evolution of electrically conductive phage-based materials

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Electrical conductivity has been observed in a few naturally-occurring bacterial protein fibers, including the pili filaments produced by Geobacter sulfurreducens, and the extracellular appendages that extend the membrane of Shewanella oneidensis bacteria. In these conductive protein materials, electron transport is thought to be mediated by a combination of intra- and intermolecular electron delocalization and electron hopping across redox centers. Biomimetic engineering has also been employed to synthesize bacterial amyloid fibers or self-assembling peptides with enhanced conductivity. However, conductivity mechanisms are difficult to elucidate in protein fibers. Aromatic, charged and polar amino acids, protein structure, and chemical environment all influence the final conductivity of protein fibers. Electronic, protonic and ionic transport can occur in these complex materials, thus making it difficult to rationally engineer synthetic protein materials with conductivity levels that surpass the naturally conductive bacterial protein fibers.In this proposed work, we unite a biomaterials engineer, a physicist and a chemical biologist to tackle an important question in protein-based electronics. Namely, we will utilize directed evolution to evolve and allow nature to guide the design process of conductive protein fibers. We will achieve this by developing a novel directed evolution scheme for evolving and isolating conductive protein fibers, and screening them for conductivity.Specifically, we will investigate the use of filamentous M13 bacteriophages as biomolecules that assemble into proteinaceous fiber-like structures and that contain the genetic material necessary to trace the evolutionary mutations acquired through rounds of directed evolution. We will apply random and site-directed saturation mutagenesis approaches on the repeated pVIII coat protein of the bacteriophage, targeting its surface- exposed N-terminal variable region that has been shown to tolerate mutations without disrupting assembly of phage particles. To screen individual phage mutants after each round of evolution, we will utilize the Convex Lens-induced Confinement (CLiC) technology to gently squeeze single bacteriophages into nanoscale compartments. Upon isolation, we will perform direct conductivity measurements using nano-patterned electrodes inside the nano-compartments, which will also serve to orient the phages. Through repeated rounds of evolution, we will analyze the combinations of residues that contribute to electrical conductivity enhancement, and will gain critical knowledge about charge transport along protein fibers. Further, by selecting the most conductive mutants, we will evolve bacteriophages that could be utilized in a range of nano-electronic devices, and to be integrated in functional devices such as biosensors, bio-electrodes, wearables, biomaterials for electrical cell and tissue stimulation and various bio-inorganic interfaces.Overall, this project will contribute to fundamentally understanding charge transport in proteins, and to developing sustainable material alternatives for next-generation bio-electronics. It will also allow for developing new methods to screen for properties that have not previously been evolved via directed evolution. Through focused training and efforts of several trainees who will be involved in this multidisciplinary project, we expect these outcomes to impact nanotechnology and biomedical industries in Quebec and Canada.