Directed Evolution of Electron Conduits

描述（由申请人提供）：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，使其与环境电隔离。这项提案的重点是利用定向进化来设计电子导管，使重组细胞能够有效地将电子穿过细胞膜。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，允许重组微生物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慢十倍。最近的研究结果表明，在重组细胞中的蛋白质间电子转移是有限的。因此，我们建议工程化的蛋白质负责的重组细胞中的瓶颈，以提高电子传递效率。具体目标是：（1）开发用于测定电子传递过程中蛋白质功能的筛选，（2）通过定向进化工程化目的蛋白质以提高电子传递效率，和（3）评价当导管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，外源性蛋白质间的相互作用限制了电子通量，该项目提出通过定向进化优化这种非自然的相互作用。这些目标将使用Frances Arnold小组提供的工具和技术，结合Caroline Ajo-Franklin小组密切合作开发的检测方法来实现。该蛋白质转移电子的能力将使用实验室开发的比色分析进行筛选。该测定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鉴定，阐明蛋白质的结构-功能关系，并帮助理解该系统中电子传递的详细机制。最后，将优化相关残基，并将修饰的蛋白质与导管共表达，以评估细胞外电子传递的改善。将使用比色测定和电极测量监测细胞外转运。

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.