Synthetic engineering of conductive biofilm development in the y-proteobacterium Shewanella oneidensis

本提案的目的是了解奈氏希瓦氏菌在电极表面形成导电生物膜的边界条件，以及生物内源性构建这些电流产生结构的高级能力的合成发展。关于细胞外电子转移和与不溶性电子受体一起茁壮成长的能力，野刺草是最容易理解的模式生物。尽管如此，与其他细胞外电子转移的模式生物-硫还原地杆菌相比，这些电子受体的可实现电子转移率要低几倍。我们的研究建立在一个假设的基础上，即赤铁矿等电子受体的减少或生物电化学系统中产生电流的能力与产生导电生物膜的能力有关。G.硫还原细胞在其细胞外聚合物质中整合导电结构的能力证实了这一点。在提出的工作中，我们将从根本上确定如何通过添加非生物或生物导电结构来控制s.o idensis在阳极表面上的生物膜生产。为此，我们将使用最近建立的用于生物电化学系统的先进微流控平台。我们想了解有机体如何对这种材料的整合做出反应，以及使用这些外源性电流收集器可以建立的最大电流密度是多少。当我们设计S. oneidensis在其细胞外聚合物材料中内源性产生导电结构时，这个最大电流密度将是我们想要达到的基准。我们将外膜细胞色素输出到基质中，并通过异肽键将它们与细胞表面的其他蛋白质合成连接。为了达到这一目标，我们将使用最近建立的SpyTag/SpyCatcher技术，该技术允许蛋白质的翻译后连接。

The aim of this proposal is to understand the boundary conditions of conductive biofilm development by Shewanella oneidensis on electrode surfaces and the synthetic development of an advanced ability of the organism to endogenously build these current producing structures. S. oneidensis is the best understood model organism regarding extracellular electron transfer and the ability to thrive with insoluble electron acceptors. Still, the achievable electron transfer rates with these electron acceptors are several fold lower compared to the other model organism for extracellular electron transfer – Geobacter sulfurreducens. Our research builds upon the hypothesis that the reduction of electron acceptors like hematite or the ability to produce a current in a bioelectrochemical system is correlated to the ability to produce conductive biofilms. This is corroborated by the ability of G. sulfurreducens cells to integrate conductive structures in its extracellular polymeric substance. In the proposed work we will fundamentally establish how biofilm production on anode surfaces by S. oneidensis can be steered by the addition of abiotic or biotic conductive structures. To this end we will use a recently establish microfluidic platform that was advanced for use with bioelectrochemical systems. We want to understand how the organism reacts to the integration of this material and what the maximum current densities are that can be established using these exogenous current collectors. This maximum current density will be the benchmark that we want to reach when we engineer S. oneidensis to endogenously produce conductive structures within its extracellular polymeric material. We will export outer membrane cytochromes into the matrix and will synthetically connect them via isopeptide bonds to other proteins on the surface of the cell. To reach this goal we will use the recently established SpyTag/SpyCatcher technology that allows the posttranslational connection of proteins.