Extracellular electron transfer (EET) pathways, such as those in the bacterium Shewanella oneidensis, interface cellular metabolism with a variety of redox-driven applications. However, designer control over EET flux in S. oneidensis has proven challenging because a functional understanding of its EET pathway proteins and their effect on engineering parametrizations (e.g., response curves, dynamic range) is generally lacking. To address this, we systematically altered transcription and translation of single genes encoding parts of the primary EET pathway of S. oneidensis, CymA/MtrCAB, and examined how expression differences affected model-fitted parameters for Fe(III) reduction kinetics. Using a suite of plasmid-based inducible circuits maintained by appropriate S. oneidensis knockout strains, we pinpointed construct/strain pairings that expressed cymA, mtrA, and mtrC with maximal dynamic range of Fe(III) reduction rate. These optimized EET gene constructs were employed to create Buffer and NOT gate architectures that predictably turn on and turn off EET flux, respectively, in response to isopropyl β-D-1-thiogalactopyranoside (IPTG). Furthermore, we found that response functions generated by these logic gates (i.e., EET activity vs inducer concentration) were comparable to those generated by conventional synthetic biology circuits, where fluorescent reporters are the output. Our results provide insight on programming EET activity with transcriptional logic gates and suggest that previously developed transcriptional circuitry can be adapted to predictably control EET flux.

中文翻译：

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使用转录逻辑门调节 Shewanella oneidensis 的细胞外电子转移。

细胞外电子转移 (EET) 途径，例如细菌Shewanella oneidensis中的途径，将细胞代谢与各种氧化还原驱动的应用相结合。然而，设计者对S. oneidensis 中EET 通量的控制已被证明具有挑战性，因为通常缺乏对其 EET 途径蛋白质及其对工程参数化（例如，响应曲线、动态范围）的影响的功能理解。为了解决这个问题，我们系统地改变了编码S. oneidensis主要 EET 途径部分的单个基因的转录和翻译，CymA/MtrCAB，并检查了表达差异如何影响 Fe(III) 还原动力学的模型拟合参数。使用由适当的S. oneidensis敲除菌株维持的一套基于质粒的诱导回路，我们确定了表达cymA、mtrA和mtrC 的构建体/菌株配对具有最大动态范围的 Fe(III) 还原率。这些优化的 EET 基因构建体用于创建缓冲和非门结构，分别响应异丙基 β-D-1-硫代吡喃半乳糖苷 (IPTG) 可预测地打开和关闭 EET 通量。此外，我们发现这些逻辑门产生的响应函数（即 EET 活性与诱导剂浓度）与传统合成生物学电路产生的响应函数相当，其中荧光报告基因是输出。我们的结果提供了对使用转录逻辑门编程 EET 活动的见解，并表明先前开发的转录电路可以用于可预测地控制 EET 通量。