Wet Lab Part

Modification of sfGFP reporter system

In order to find out the sgRNA that can repress PilA expression in Vibrio natriegens best, we designed a reporter system based on sfGFP. A perfectly working CRISPRi system can elevate the ratio of our target Geobacter metallireducens pilus in the final product and simplify the harvest step.

为了找到抑制PilA表达效果最好的sgRNA,我们设计了一个基于sfGFP的报告系统。一个完美工作的CRISPRi系统能够提高我们的目标——金属还原地杆菌菌毛在产物中的占比并且简化菌毛收获步骤。

Cycle 1 The first version of report plasmid pGFP

design

In the first reporter plasmid pGFP, the expression of sfGFP is controlled by the promoter of V.natriegens PilA and a small part of PilA is merged to the N terminus of sfGFP(fig.1). The target sequences of the four candidate sgRNAs we initially chose reside in this region.

在第一个报告质粒pGFP中，sfGFP的表达由需钠弧菌PilA基因的启动子控制，并且sfGFP的末端融合了一小段PilA序列（图1）。我们最初选择的四个候选sgRNA的目标序列都位于这个区域之内。

Build

pGFP was ordered from GENWIZ. The structure of the plasmid is illustrated below(fig.2)

pGFP质粒是从GENWIZ订购的。该质粒的结构如下所示（图2）。

test

We tried to measure fluorescence intensity using a microplate reader, but we failed to detect difference between the transferomed and not transformed V.natriegens. Then we tried SDS-PAGE to check whether the sfGFP was successfully experssed. There was a band between 35kD and 25kD(the molecular weight of recombined sfGFP is about 30kD) in the lane of soluable proteins extracted from V.GF4(ATCC 14048 transfomred with pGFP) and not in the lane of proteins from the wild type strain(fig.3). The SDS-PAGE was repeated three times to validate the result.

我们尝试使用酶标仪来测量荧光强度，但在转化和未经转化的需钠弧菌中并没有检测到荧光强度的差异。之后我们尝试用SDS-PAGE来检查dfGFP是否成功表达。从V.GF4（转入sfGFP的ATCC 14048菌株）中提取的可溶性蛋白经电泳后在35kD和25kD之间有一个条带（重组sfGFP的分子量约为30kD），而野生型菌株对应的泳道中无相应条带。

learn

The result of SDS-PAGE showed that the recombinant sfGFP was expressed in V.GF4. But maybe due to the PilA merged to the N terminus of sfGFP, it could not fold properly, so no fluorescence signal was detected.

SDS-PAGE的结果表明V.GF4中表达了重组sfGFP。但可能是由于sfGFP N末端融合的PilA片段，sfGFP无法正常折叠，因而无法检测到荧光信号。

After consulting professor Guo, we knew that the signal sequence in the merged part might interfere with the folding of sfGFP because this sequence can interact with other proteins. Besides, sfGFP can fold properly when the merged part is significantly larger or smaller than itself, or when there is a linker sequence between the two parts. However, the lengths of the merged PilA and sfGFP were similar.

在请教郭教授后，我们了解到融合部分的信号序列会与其他蛋白质相互作用，因而可能会干扰sfGFP的折叠。此外，当添加的片段大小显著大于或小于sfGFP的大小时，或当被融合的两部分之间存在连接序列时，sfGFP也能够正常折叠。然而，我们所添加的PilA片段的长度与sfGFP近似。

Cycle 2 The second version of reporter plasmid

design

In consideration of the possible problems we identified, we designed a second version of reporter plasmid. We planned to delete the PilA part in the reporter plasmid so that there remained only two sgRNA target sites in this version of plasmid(fig.4).

考虑到已被发现的一些可能的问题，我们设计了第二版报告质粒。我们计划删除报告质粒中的PilA片段，所以在这版质粒中知保留了两个sgRNA位点。

Build

We plan to modificate pGFP to get the new version of reporter plasmid, pGFPcut, by reverse PCR and Gibson assembly(fig.5). Homologous arm was added to one of the primers for reverse PCR. The result plasmid pGFPcut was first transformed into DH5α and verified by DNA sequencing.

我们计划通过反向PCR和Gibson assembly来改进pGFP，得到新版的报告质粒pGFPcut。在其中一个引物上添加了同源臂以进行反向PCR。得到的pGFPcut质粒首先被转入DH5α中并通过DNA测序进行验证。

learn

Although pGFPcut can express properly folded sfGFP as a reporter gene in V.natriegens and contains two sgRNA target sites, we still wanted to test the other two sgRNAs.

尽管pGFPcut能够在需钠弧菌中表达正确折叠的sfGFP作为报告基因，并且包含了两个sgRNA位点，我们仍然想要进一步测试另外两个sgRNA。

cycle 3 The third version of report plasmid

design

In order to test the other two sgRNAs, we decided to further improve the reporter plasmid. Based on the first version, pGFP, we plan to alter the position of the initiation coden so that the PilA part could not be translated(fig.6). In this new version, the three sgRNAs can be more precisely tested.

为了测试另外两个sgRNA，我们决定进一步改进报告质粒。基于第一版质粒pGFP，我们计划改变起始密码子的位置，使得PilA片段无法被翻译。在这一新版本中，候选的三种sgRNA可被更加精确地测试。

Build

This time the modification was also based on mutagenesis PCR and Gibson assembly(fig.7). We used two pairs of primers to change the two methionines to threonine. The resulting plasmid pGFPmod1 and pGFPmod2 were verified by DNA sequencing.

这一次的改进也是基于定点突变PCR和Gibson assembly。我们使用两对引物分别将两个甲硫氨酸突变为苏氨酸。得到的质粒pGFPmod1和pGFPmod2通过DNA测序进行验证。

learn

pGFmod2 was successfully transfered into wild type Vibrio natriegens as verified by colony PCR. V.natriegens transformed with pGFPmod2 and pGFPcut were named as V.mod and V.cut respectively. The fluorescence intensity was measured using microplate reader and the result was illustrated below(fig.8). Three parallel tests were conducted for the three strains and the average index of blank control(LB+v2 salts only) was deducted from original data. The result showed that V.mod and V.cut had stronger fluorescence than the wild type strain.

pGFmod2被成功转入野生型需钠弧菌中并且经菌落PCR验证。将转入pGFPmod2和pGFPcut的需钠弧菌分别命名为V.mod和V.cut。用酶标仪测定荧光强度，结果如下（图8）。三个菌株各进行了三组平行实验。空白对照（LB+v2 salts）的平均值由原始数据推出。结果显示V.mod和V.cut的荧光强度强于野生型。

Pili harvest

Cycle 1 shear and filter

design

In the early stage of our project design, we planned to use the method of shearing by blender and filteration to harvest e-pili(fig.9). This method of pili harvest was originally used to harvest pili of Geobactor sulfurreducens and two previous iGEM teams(GreatBay_SZ 2020 and LINKS_China 2020) used this method before.

在项目设计的早期，我们计划使用搅拌机搅碎并过滤的方式来收获e-pili（图9）。这种菌毛收获方法最初被用于收获硫地还原杆菌的菌毛。先前有两支iGEM队伍(GreatBay_SZ 2020 and LINKS_China 2020) 使用过该种方法。

Test

We tried to harvest our e-pili according to this protocol see experiment. Generally, the cells were scraped from plates and collected with LB+v2 salts medium and then pelleted by centrifugations and resuspended in ethanolamine buffer. The mixture was blended to shear pili from cell surfaces and cell debris were removed from the mixture. Finally, the solution was filtered with a 100 kDa-molecular-weight cutoff PVDF membrane. However, when testing the resistance of protein membrane made of harvested sample solution, we found that the resistance was too high(about 0.7 MΩ). So we tried transmission electron microscope to see microstructure of this solution and the result was shown in fig.10. There were many thick fibers in the sample which were not e-pili.

我们尝试使用以下实验步骤来收获e-pili。简单来说，就是从平板上刮下细菌，收集在LB+v2 salt培养基中。之后离心，去上清，并用乙醇胺缓冲液重悬沉淀。得到的混合物加入搅拌机中搅拌，菌毛从细胞表面被剪切下来，细胞碎片被除去。最后，用PVDF-100kDa超滤膜过滤溶液。然而在测试由收获的样品溶液制成的蛋白质膜的电阻时，我们发现其电阻过高（约0.7 MΩ）。所以我们用透射电子显微镜对溶液的显微结构进行了观察，结果如图10所示。在样品中可见很多并非e-pili的粗纤维。

learn

We inferred that these fibers may be scrapped from PVDF membrane surface when collecting e-pili in the final step of pili harvest. However, this protocol of pili harvest had been used by other investigators[1] and iGEM teams. The problem may be caused by the difference in filter membrane brands and we might wash the membrane too many times at the final step.

我们推测这些纤维可能是在收获菌毛的最后一步中从PVFD膜表面上被刮下的。然而，这一收获菌毛的实验步骤曾成功地被其他研究者和iGEM队伍使用过。问题可能出在超滤膜品牌的不同，也可能是由于我们在最后一步中将滤膜清洗了太多次。

Cycle 2 shear and precipitate

design

After reading more relevant articles, we found another method to harvest pili. This method was similar to the first one, but after shearing, cell debris was removed from the solution by centrifugation. Then, e-pili was collected by ammonium sulfate precipitation(fig.11).

在阅读了更多相关文章后，我们找到了另一种收获菌毛的方法。这一方法与之前的类似，不同之处在于搅碎之后的细胞碎片通过离心从溶液中被移除。然后再通过硫酸铵沉淀法收集e-pili。

test

TEM and resistance measurement were also performed using purified pili solution harvested using this method. This time the resistance of the protein membrane was much lower, which was about 17 kΩ. The result of TEM showed that there were several nanowires in the solution, which we inferred to be the e-pili we wanted.

对用这种方法收获的菌毛溶液也进行了电镜观察和电阻测量。这一次蛋白质膜的电阻与之前相比显著降低，约为17 kΩ。电镜观察结果显示，溶液中存在着一些纳米线，我们推测这些纳米线就是我们想要得到的e-pili 。

learn

Using the second method, we successfully acquired pili slution that had much lower resistance than that of 5% NaCl solution(see result ). However, the resistance of this pili was still higher than formerly reported[2]. The reason for this result might be that there were still many impurities in our sample due to insufficient speed provided by our centrifuge when removing debris from the pili-solution. Also, we are not skilled in purification with Ni-NTA resin and sample preparation for TEM.

使用第二种收获方法，我们成功得到了电阻率显著低于5% 氯化钠溶液的菌毛溶液。然而，该菌毛溶液的电阻仍高于先前的相关报告。这一结果的原因可能是：将细胞碎片菌毛溶液中去除时，由于我们的离心机转速不够，导致我们的最终样品中仍然存在很多杂质。同时我们对于使用Ni-NTA树脂纯化蛋白和准备电镜观察样品也不够精通。

Hardware Part

Bio-degradable Electrode for E-Pili Cartridge

As E-Pili Cartridge is produced, used and discarded by the millions, and its use is no longer limited to healthcare facilities, we believe it is important to minimize the use or percentage of unsustainable components in this consumable. Considering the SDGs of poverty eradication and responsible consumption and production: we believe that the use of unsustainable components or the percentage of such components in this consumable should be minimized in order to reduce the damage to the environment throughout the entire life cycle of the consumable. And to provide the lowest possible cost options for E-Pili Cartridge to make them affordable to a wider range of potential users (outside of urban community health systems).

由于E-Pili Cartridge会数以千万记地被生产、使用和丢弃以及使用场景不再局限在医疗机构内，考虑到可持续发展目标中消除贫困和负责任地消费和生产：我们认为应当尽可能减少这种耗材中不可持续的成分使用或该成分的占比，以减少耗材的全生命周期对环境的损害；并提供尽可能低成本的耗材方案，以让更多的潜在用户（城市社区卫生体系以外）得以负担得起。

design

Structures in the conventional lateral flow immunochromatographic assay include: NC membrane, absorbent pad, sample pad, PVC base plate and casing, to which our design adds E-Pili and electrodes. The parts of the Cartridge that are difficult to degrade are the electrodes, the PVC base plate and the casing. Since the rapid test consumables with electrodes are the more innovative part of our design, we wanted to focus on responsible production by replacing the more commonly used copper electrodes with biodegradable electrode materials; there are already examples of pulp-based materials used in the base plate and casing, so we are not exploring this part of the design for the time being.

常规的侧流免疫层析测定方式中的结构包括：NC膜、吸水垫、样品垫、PVC底板和外壳，我们的设计在其中添加了菌毛和电极。耗材中难以降解的部分有电极、PVC底板和外壳。由于带有电极的快速检测耗材是我们设计中较为创新的部分，我们希望将负责任的生产集中落实于此，用可降解的电极材料替换更常用的铜电极；底板与外壳已有纸浆基材料制造的案例可参考，故我们暂不在这个部分做探索。

During our research we found a HCI paper that describes sustainable electronic prototyping with bio-based and biodegradable materials, providing formulations for conductive bioplastics in the form of sheets, pastes and foams. We attempted to explore molding methods using the sheet and paste forms of this conductive bioplastic as materials to be used as electrodes for the E-Pili Cartridge.

在调研中我们发现了一篇人机交互论文，论文介绍了用以生物基且可生物降解的材料进行可持续电子原型制作方式，提供了片材、糊剂和泡沫三种形式的导电生物塑料的配方。我们试图用这种导电生物塑料的片材和糊剂形式为材料，探索成型方式，将其作为E-Pili Cartridge的电极。

Build

We experimented with electrode materials at Fablab Shanghai and tried both sheet and paste forms of them as fabricated in the paper. The Carboxymethyl Cellulose (CMC) based paste was very prone to drying and cracking and could not be molded, so no further attempts were made.

我们在Fablab Shanghai进行电极材料的实验，并按照论文中的制作方式尝试了其中的片材和糊剂两种形式，其中羧甲基纤维素（CMC）为基材的糊剂十分容易干裂，无法成型，故未做进一步尝试。

Sheets were made with 3g of sodium alginate as a base, mixed with 75g of distilled water and 3ml (3.8g) of glycerol was added. After standing for two hours, 1.95g of carbon black powder was added and stirred. The mixture was applied to an acrylic plate (the plate brush was set up on a 2mm thick strip of cork to control the thickness), to which a 10% calcium acetate solution was sprayed. It was then dried intermittently in an oven at 50°C for two days (with the oven door slightly open to prevent overheating and warping).

片材以3g海藻酸钠为基底，与75g蒸馏水混合，加入3ml（3.8g）甘油。静置两小时后，加入1.95g炭黑粉末搅匀。将混合物涂布在亚克力板上（将板刷架在2mm厚的软木条上以控制厚度），向其上喷洒10%醋酸钙溶液。随后放入烤箱50℃间断地烘干两天（烤箱门稍开，防止过热翘边）。

test

A variety of ratios and drying methods were tried and documented. The resistance of the sheet with sodium alginate as the base material and carbon black as the conductor was large (on the order of 10 kΩ in the initial experiment), but after raising the proportion of carbon black (3.5 g) and reducing the proportion of glycerol (3.5 g), the smallest portion of the sheet resistance (with the meter pens spaced at 1 cm apart) could be controlled to be in the range of 20-60Ω.

我们进行了多种配比和干燥方式的尝试，并将其记录下来。海藻酸钠为基材、炭黑为导电成分的片材电阻较大（初次实验为10kΩ数量级），但在提升炭黑比例（3.5g）并减少甘油比例（3.5g）后，片材电阻最小的部分（表笔间距1cm)可以控制在20-60Ω。

learn

We obtained materials with more ideal resistance values by changing the material ratio, but the way of producing such bio-based electrodes in Fablab is rather rough. Under the current experimental conditions, different batches of materials are affected by humidity, area, substrate/mold, etc., and the resistance and flatness of the output materials are not guaranteed to be stable. We tried to compensate for the effect of electrode instability by first measuring the electrode resistance, then the resistance of electrode + E-Pili, and finally calculating the difference to get the resistance of E-Pili. The distribution of resistance in a piece of material was also not uniform. It is noteworthy that we found that the resistance of the part of the material that was tightly attached to the acrylic plate after drying and smooth (visibly reflective) when removed was very small (around 20 Ω), whereas the resistance of the large, rough, and dry part was still very large (on the order of 100-1000 Ω).

我们通过改变材料比例获得了电阻值较为理想的材料，但在Fablab中生产这种生物基电极的方式比较粗糙，目前实验条件下，不同批次材料受湿度、面积、基底/模具等因素的影响较大，产出材料的电阻、平整程度都无法保证稳定。我们试图用先测定电极电阻，再测电极+菌毛的电阻，最后计算差值得到菌毛电阻的方式来补偿电极不稳定的影响。一块材料中的电阻分布也并不均匀。值得注意的是我们发现烘干后紧贴亚克力板，摘下后光滑（明显反光）的部分电阻很小（20Ω左右），而大面积的粗糙且干燥的部分电阻仍很大（100-1000Ω数量级）。

Another issue of great impact is that this sheet material cannot be manufactured as finely as metal for the time being in the form of interdigital electrodes, nor can it be molded into cylinders to serve as contacts between the Cartridge and Plinker.

另一个影响很大的问题是这种片状的材料暂时无法像金属一样很精细地加工为叉指电极的形式，也很难成型为圆柱体作为耗材和设备之间的触点。

The question of how to process this material so that it can be used as our electrodes is an open one, but we have had to put the exploration of sustainable electrodes on hold due to the project timeline. However, we will be actively exploring ways in which it can be stabilized and manufactured in a way that makes our Cartridge environmentally friendly.

对于这种材料如何加工以使其可以用作我们的电极是一个有待解决的问题，但由于项目周期问题我们不得不搁置对于可持续电极的探索。但我们会积极探索使其可以被稳定制造的方式，让我们的耗材对环境友好。

Engineering cycle

cycle1

Design

During the initial debugging phase, we conducted a study on the expansion of WiFi functionality for the STM32. The basic process for testing the WiFi functionality of the STM32 chip includes importing relevant serial port libraries, using the WiFi module for communication tests to achieve data reception and transmission.

Build

We performed WiFi debugging using the STM32 chip. Firstly, we imported the serial port library "serial" in Python to read the serial port data. Upon successful reading, since the transmitted signal is in the form of a floating-point number with 8 significant digits, we needed to convert the string data into an integer and set the decimal places accordingly.

Test

We conducted communication testing using the WiFi module of the STM32 chip. First, we ensured the WiFi module was properly connected to the WiFi network we set up and connected it based on the account and password we set. Then, we sent WiFi connection instructions to the WiFi module via serial communication to ensure connection with the WiFi network. If the connection was successful, the WiFi module would return connection status information and we could use it for data transmission.

Learn

Through this study, we learned the basic process and debugging methods of using the WiFi function of the STM32 chip. We mastered skills such as serial data reading and sending connection instructions to the WiFi module. At the same time, we also deepened our understanding of the WiFi function in chip design, which is very helpful for subsequent device design and development.

cycle2

Design

Through this study, we learned the basic process and debugging methods of using the WiFi function of the STM32 chip. We mastered skills such as serial data reading and sending connection instructions to the WiFi module. At the same time, we also deepened our understanding of the WiFi function in chip design, which is very helpful for subsequent device design and development.

Build

We establish a real-time curve plotting model in the form of dynamically displaying a conductivity curve chart. In Python, we use library functions for graphical plotting. Firstly, we use the functions under the library to set the initial set and use pyqtgraph to draw the initial curve. Once started, the serial port begins receiving data. Each time new data is received, it is added directly to the set, and the entire curve is updated simultaneously, achieving real-time curve plotting. In order to achieve the effect of real-time display, after each new data is added to the end of the set, the overall image needs to be shifted left by one data, that is, the beginning data in the set is deleted, and the entire curve is redrawn.

Test

Learn

We learned how to use Python to realize real-time curve plotting of data, as well as the skill of connecting with STM32 and reading data. We mastered the basic process of using the matplotlib library and pyqtgraph library to plot curves, and understood the importance of filtering and data processing. In addition, we also learned how to set appropriate parameters and achieve real-time plotting to ensure the accuracy and readability of curve plotting.

cycle3

Design

Finally, we debugged the STC15 chip and ESP8266 WiFi module, which are closer to the functionality of conductivity testing, for hardware connection. We used serial communication functions to communicate and configure the ESP8266 module, and ultimately ensured that the ESP8266 module can communicate normally between the client and server side.

Build

First, connect the hardware equipment, including the ESP8266 WiFi module, STC15 chip development board, and module for testing data. Connect the TXD pin of the ESP8266 module to the RXD pin of the STC15 development board, and connect the RXD pin of the module to the TXD pin of the development board. Also, connect the VCC pin to the 5V power pin on the development board and the GND pin to the GND pin on the board. Next, use a USB to TTL serial module to connect the STC15 development board to the computer.

During serial communication, use the sending and receiving commands in the STC15 development environment to communicate with the ESP8266 module. After setting the AT command, send the AT command through UART to set up the ESP8266 module, including checking its normal operation and setting up WiFi connection parameters. To ensure that the ESP8266 module works properly, write code using serial communication functions.

During serial communication, use the sending and receiving commands in the STC15 development environment to communicate with the ESP8266 module. After setting the AT command, send the AT command through UART to set up the ESP8266 module, including checking its normal operation and setting up WiFi connection parameters. To ensure that the ESP8266 module works properly, write code using serial communication functions.

Test

When conducting client testing, the first step is to download a TCP client software and enter the IP address and port number of the ESP8266 module in the software for connection testing and message sending and receiving testing. Throughout the testing process, it is important to ensure that the network environment is stable and that the WiFi name and password set in the software are correctly connected. Before starting the test, check the connection status of the ESP8266 module, such as the flashing of the indicator lights, to ensure that a proper connection can be established. During testing, it is necessary to pay attention to the stability of the communication and to conduct a long-term testing to ensure stable data transmission. By conducting client testing, it further verifies that the STC15 chip and ESP8266 module can work properly and ensure smooth communication under all circumstances.

Learn

We have learned how to debug hardware devices, communicate using serial communication functions, and perform client testing. We also learned about the connection methods between the STC15 chip and the ESP8266 WiFi module, which can improve performance in applications like conductivity testing. In addition, we learned how to ensure network stability and reliable data transmission for more reliable communication in practical applications.