Directed evolution of electrically conductive phage-based materials

导电性已在一些天然存在的细菌蛋白纤维中被观察到，包括硫还原地杆菌产生的毛细丝，以及延伸希瓦氏菌膜的细胞外附属物。在这些导电蛋白材料中，电子传递被认为是由分子内和分子间的电子离域和氧化还原中心的电子跳变共同介导的。仿生工程也被用于合成具有增强导电性的细菌淀粉样纤维或自组装肽。然而，蛋白质纤维的传导机制很难阐明。芳香、带电和极性氨基酸、蛋白质结构和化学环境都会影响蛋白质纤维的最终电导率。电子、质子和离子传输可以发生在这些复杂的材料中，因此很难合理地设计出具有导电性水平超过天然导电性细菌蛋白质纤维的合成蛋白质材料。在这项拟议的工作中，我们联合了一位生物材料工程师，一位物理学家和一位化学生物学家来解决蛋白质电子学中的一个重要问题。也就是说，我们将利用定向进化来进化，让自然来指导导电蛋白纤维的设计过程。我们将通过开发一种新的定向进化方案来进化和分离导电蛋白纤维，并筛选它们的导电性来实现这一目标。具体来说，我们将研究丝状M13噬菌体作为生物分子的使用，这些生物分子组装成蛋白质纤维样结构，并包含追踪通过轮次定向进化获得的进化突变所需的遗传物质。我们将对噬菌体的重复pVIII外壳蛋白应用随机和定点饱和诱变方法，靶向其表面暴露的n端可变区域，该区域已被证明能够耐受突变而不破坏噬菌体颗粒的组装。为了在每一轮进化后筛选单个噬菌体突变体，我们将利用凸透镜诱导约束（CLiC）技术将单个噬菌体轻轻挤压到纳米级的隔室中。分离后，我们将在纳米隔间内使用纳米图案电极进行直接电导率测量，这也将用于定向噬菌体。通过反复的进化，我们将分析有助于电导率增强的残基组合，并将获得关于蛋白质纤维电荷传输的关键知识。此外，通过选择最导电的突变体，我们将进化出可用于一系列纳米电子器件的噬菌体，并将其集成到功能器件中，如生物传感器、生物电极、可穿戴设备、用于细胞和组织刺激的生物材料以及各种生物无机界面。总的来说，这个项目将有助于从根本上理解蛋白质中的电荷传输，并为下一代生物电子学开发可持续的材料替代品。它还将允许开发新的方法来筛选以前没有通过定向进化进化的属性。通过将参与这个多学科项目的几名受训者的重点培训和努力，我们期望这些成果能够影响魁北克和加拿大的纳米技术和生物医学工业。

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.