Directed evolution of electrically conductive phage-based materials

在一些自然产生的细菌蛋白纤维中观察到了导电性，包括硫还原地杆菌产生的菌毛细丝，以及延伸伊氏希瓦氏菌膜的胞外附件。在这些导电蛋白质材料中，电子传递被认为是通过分子内和分子间的电子离域以及跨氧化还原中心的电子跳跃来调节的。仿生工程也被用来合成具有增强导电性的细菌淀粉样纤维或自组装肽。然而，蛋白质纤维的导电机制很难阐明。芳香族氨基酸、带电氨基酸和极性氨基酸、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。在这些复杂的材料中可以发生电子、质子和离子的传输，因此很难合理地设计出具有超过自然导电细菌蛋白纤维的电导率的合成蛋白质材料。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基于蛋白质的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，并让自然来指导导电蛋白质纤维的设计过程。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，从而实现这一点。具体地说，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，并包含追踪通过几轮定向进化获得的进化突变所需的遗传物质。我们将对噬菌体重复的pVIII外壳蛋白应用随机和定点饱和突变方法，针对其表面暴露的N末端可变区，该可变区已被证明在不破坏噬菌体颗粒组装的情况下耐受突变。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限制(CLIC)技术将单个噬菌体轻轻挤压到纳米级的隔间。在分离后，我们将使用纳米隔间内的纳米图案电极进行直接电导率测量，这也将用于定位噬菌体。通过重复几轮的进化，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，通过选择导电性最强的突变体，我们将进化出可用于一系列纳米电子设备的噬菌体，并将其集成到生物传感器、生物电极、可穿戴设备、用于电细胞和组织刺激的生物材料以及各种生物-无机接口等功能设备中。总的来说，该项目将有助于从根本上了解蛋白质中的电荷传输，并为下一代生物电子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特性。通过集中培训和将参与这一多学科项目的几名学员的努力，我们预计这些成果将影响魁北克和加拿大的纳米技术和生物医药行业。

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.