Directed Evolution of Electron Conduits

描述(申请人提供)：下一代生物传感和生物医学设备需要细胞与合成结构(如电极)电连接。然而，细胞被绝缘膜包围，这些绝缘膜将它们与环境电隔离。这项提议的重点是使用定向进化来设计电子管道，使重组细胞能够有效地将电子传输到细胞膜上。最近的一项研究在大肠杆菌中异源表达了三种蛋白质，MTRA、mtrB和mtrC，使重组微生物能够通过其绝缘膜传输电子。与通过mtrABC途径输出电子的自然系统相比，重组细胞输出电子的速度要慢十倍。最近的研究结果表明，重组细胞中蛋白质间的电子转移是有限的。因此，我们建议对重组细胞中负责瓶颈的蛋白质进行工程处理，以提高电子转移效率。其具体目标是：(1)开发一种检测蛋白质在电子转移过程中的功能的筛选；(2)通过定向进化来工程目的蛋白质以提高TS电子转移效率；以及(3)评估当管道与进化的蛋白质共表达时，细胞外电子通量是否得到改善。目前的数据表明，外源蛋白质间的相互作用正在限制电子通量，该项目建议通过定向进化来优化这种非自然的相互作用。这些目标将使用弗朗西斯·阿诺德小组可用的工具和技术，并与卡罗琳·阿乔-富兰克林小组密切合作开发的分析相结合来实现。这种蛋白质传递电子的能力将使用实验室开发的比色分析方法进行筛选。这项检测将用于筛选突变蛋白质文库。负责调节蛋白质活性的残基将被识别，阐明蛋白质的结构-功能关系，并有助于了解该系统中电子传递的详细机制。最后，相关残基将被优化，修饰后的蛋白质将与管道共表达，以评估细胞外电子传递的改善。细胞外转运将使用比色法和电极测量进行监测。

DESCRIPTION (provided by applicant): The next generation of biosensing and biomedical devices requires cells to electrically interface with synthetic constructs, such as electrodes. However, cells are surrounded by insulating membranes that electrically isolate them from their environment. This proposal focuses on using directed evolution to engineer electron conduits that will allow recombinant cells to efficiently transport electrons across their membranes. A recent study has heterologously expressed three proteins, mtrA, mtrB, and mtrC, in Escherichia coli, allowing the recombinant microbe to transport electrons across its otherwise insulating membrane. Compared to natural systems that export electrons through the mtrABC pathway, the recombinant cells export electrons ten times slower. Recent findings suggest that inter-protein electron transfer in the recombinant cell is limiting. Consequently, we propose engineering the protein responsible for the bottleneck in the recombinant cell to improve the electron transfer efficiency. The specific aims are: (1) to develop a screen for assaying protein functionality in the electron transfer process, (2) to engineer the protein of interest to improve ts electron transfer efficiency via directed evolution, and (3) to evaluate whether extracellular electron flux is improved when the conduit is co-expressed with the evolved protein. Current data suggest that exogenous inter-protein interactions are limiting the electron flux, and this project proposes optimizing this unnatural interaction via directed evolution. These aims will be addressed using the tools and techniques available in the Frances Arnold group combined with assays developed by the Caroline Ajo-Franklin group in a close collaboration. The protein's ability to transfer electrons will be screened using a colorimetric assay that has been developed in the lab. This assay will be used to screen a library of mutant proteins. Residues responsible for modulating protein activity will be identified, elucidating the structure-function relation of he protein and helping understand the detailed mechanism of electron transport in this system. Finally, the relevant residues will be optimized, and the modified protein will be co-expressed with the conduit to evaluate improvements in extracellular electron transport. Extracellular transport will be monitored using both a colorimetric assay and electrode measurements.