Why E-pili?

Geobacter is a class of rod-shaped Gram-negative anaerobic bacteria belonging to the δ-Proteobacteria class [1-3]. They are important dissimilatory iron-reducing bacteria [4]. In order to survive in harsh anaerobic environments, certain species of Geobacter can utilize extracellular metal ions as electron acceptors for respiration, maintaining their own metabolism. This extracellular electron transfer process relies on a unique type of conductive appendage known as "electrically conductive pili"[5]. Electrons are transmitted through fibrous conductive structures on the cell surface, often with nanoscale diameters but lengths extending to the micrometer range, capable of facilitating electron transfer[6,7].

地杆菌（Geobacter）是一类呈棒状的革兰氏阴性厌氧细菌，属于δ-变形菌纲[1-3],是一种重要的异化三价铁还原菌[4]。为了在严苛的缺氧环境中生活，一些种类的地杆菌可以利用细胞外的金属离子作为电子受体进行呼吸，维持自身的新陈代谢。而这种细胞外的电子传递过程需要一种特殊的导电菌毛来介导[5]。电子通过细胞表面具有导电能力的纤维状附着物进行传递.这些纤维的直径往往只有纳米级别，但是长度却可以达到微米级别，并拥有传递电子的能力，因而被命名为导电菌毛（E-pili）[6,7]。

For instance, the type IV pili of Geobacter metallireducens exhibit an exceptional electrical conductivity of up to 277 S/cm at pH 7 [8], making them the most conductive biological nanomaterials known to date. Within these conductive bacterial nanowires, there is a high density of aromatic groups, and the overlapping Π-Π orbitals of aromatic moieties imparts metallic conductivity to these organic materials [9].

例如金属还原地杆菌的Ⅳ型菌毛在pH=7时的电导率高达277S/CM[8]，是人类目前所发现的电导率最高的生物纳米材料。这种导电菌毛内部分布着高密度的芳香族基团，而这些芳香族基团的轨道重叠可以赋予这些有机材料类似金属的导电性[9]。

The e-pili we've chosen to use is derived from Geobacter metallireducens, which were discovered more recently. The latter demonstrates incredibly high electrical conductivity. This exceptionally high conductivity aligns well with the requirements of developing our high-precision quantitative detection system.

我们所选用的导电菌毛为来源于金属还原地杆菌的菌毛，后者作为一种近期被发现的导电菌毛所表现出的超高的电导率能够很好的适配我们开发高精度定量检测系统的需求。

This type of material is considered a revolutionary "green" nanomaterial. The energy required to produce these protein nanowires through microbial processes is over two orders of magnitude less than the energy needed to produce an equivalent mass of silicon or carbon nanowires. Moreover, the production process does not generate any toxic chemicals [10]. Additionally, this material is relatively robust while being biodegradable and exhibits excellent biocompatibility [11].

这种导电菌毛被认为是一种革命性的“绿色”纳米材料。用微生物生产这种蛋白质纳米线所需的能量比生产同质量硅纳米线或者碳纳米线要少两个数量级以上,而且生产的过程中不会产生任何有毒害作用的化学物质[10]。除此之外，这种材料在相对坚固耐用的同时可以被生物降解，并且具有非常不错的生物相容性[11]。

What truly inspires us among these features is its high sensitivity of electrical conductivity to surface charge distribution. Through literature research, we've found that protonation can significantly enhance the nanowires' conductivity. For instance, the conductivity of the e-pili of Geobacter sulfurreducens increased by 100 times when the pH was lowered from 10.5 to pH 2 [12]. Additionally, researchers have investigated the effects of protein tag modifications on the conductivity of nanowires at the monomer's C-terminal. The results showed that protein tag modifications (such as His tag and HA tag) did not affect the nanowires' conductive capability, but did induce changes in their conductivity [13].

而在这些特性中真正启发我们的是它的电导率对于其表面电荷的分布情况高度敏感。通过文献调研，我们发现质子化可以大幅度的提高菌毛的电导率，例如当pH值从10.5降低到pH值2时硫还原菌菌毛的电导率增加了100倍[12]。除此之外，有研究人员测试了在菌毛单体的C端进行蛋白标签修饰对其电导率的影响，最后发现蛋白标签修饰（例如His-tag和HA-tag）不会影响菌毛的导电能力但会使其的电导率发生变化[13]。

Inspired by the properties mentioned above and in conjunction with approaches taken by other research teams in developing detection systems using this material [14,15], we have modified the C-terminal of the pilus and this modification allows for specific binding to the target antibodies. While surface adsorption often induces changes in surface charge, what we need to do is to detect the changes in its electrical conductivity. Finally, by establishing a standard curve, we can achieve real-time quantitative detection of the target antibody.

通过以上两点性质的启发并结合一些其他团队利用这种材料开发检测系统的方案[14,15]，我们今年选择通过对菌毛的C端进行修饰的使其能够特异性结合待测的物质，而表面吸附物通常会引起表面电荷的变化，我们只用去检测因为这种表面电荷变化而产生的电导率的变化，结合实现测定的标准曲线就能实现对于待测物质的即时定量检测。

The Conductivity Matters!

In our system, the conductivity of the e-pili is the most important parameter. Higher conductivity allows our detection system to become more stable and accurate. Additionally, higher conductivity may also imply more possibilities for future applications of this bio-nanomaterial.

在我们的系统中，e-pili的导电性是最重要的参数。更高的导电性使我们的检测系统变得更加稳定和准确。此外，更高的导电性也可能意味着这种生物纳米材料在未来应用中的更多可能性。

At the very beginning, we wanted to improve the conductivity of the e-pili by modifying the amino acid sequence of it and to optimize the electron transport chain.To reduce the computational workload in modeling, a common practice is to simplify the model by introducing assumptions. For example, when determining constraints for optimizing, we approximate the centroid coordinates of the newly added aromatic ring by using the average coordinates of all atoms in the side chain at the modified position(To learn more: Model).However, introducing assumptions means that our model can not achieve the level of precision required in rational protein design. We can only identify several potential mutation sites, and we cannot guarantee that the final structure of the pili and the internal electron transfer will conform to our design.

最初，我们希望通过修改e-pili的氨基酸序列来提高其导电性，并优化电子传输链。为了减少建模中的计算工作量，常见的做法是通过引入假设来简化模型。例如，在确定优化约束时，我们通过使用在修改位置的侧链中所有原子的平均坐标来近似新添加的芳香环的质心坐标（了解更多：模型）。然而，引入假设意味着我们的模型无法达到理性蛋白设计所要求的精度水平。我们只能确定几个潜在的突变位点，而不能保证毛细管的最终结构和内部电子转移会符合我们的设计。

This situation is quite common in protein structure optimization, and the most common solution to address this is to use a non-rational (high-throughput screening) approach to compensate for the shortcomings of rational design.

这种情况在蛋白质结构优化中很常见，解决这个问题的最常见方法是使用非理性（高通量筛选）方法来弥补理性设计的不足。

Directed evolution simulates the process of Darwinian evolution in the laboratory by creating a large number of mutants through random mutation and recombination. Specific selection pressures are applied for desired features, allowing the selection of proteins with desired characteristics, achieving molecular-level simulated evolution.

定向进化在实验室中模拟达尔文进化过程，通过随机突变和重组创建大量突变体。对所需特性施加特定的选择压力，允许选择具有所需特性的蛋白质，实现分子级模拟进化。

Among the various methods of directed evolution, semi-rational directed evolution is the most widely applied with the most successful cases. This method aims to mutate proteins based on some understanding of their physicochemical properties, three-dimensional structure, structure-activity relationships, catalytic mechanisms, etc. With the assistance of computers, it then uses a reasonable high-throughput screening method to quickly obtain the target mutants.

在各种定向进化方法中，半理性定向进化是应用最广泛、成功案例最多的。该方法旨在基于对蛋白质的物理化学性质、三维结构、结构-活性关系、催化机制等的一些了解来突变蛋白质。在计算机的帮助下，然后使用合理的高通量筛选方法快速获得目标突变体。