p-Cresol is an organic compound that is widely used in industry. It can be used to make a variety of chemical products and materials, such as fragrances and fragrances, dyes and softeners, certain pharmaceuticals and medical products, biocides and antiseptics, paints, industrial cleaners, etc. However, it should be noted that p-cresol has a certain degree of toxicity, and its industrial use requires strict safety operations and control measures to protect the health and safety of workers and the environment. At present, factories mainly use methods such as gas detection instruments, air sampling and analysis, color indicators, online monitoring systems, and wearable sensors to monitor p-cresol. However, these methods have certain flaws, such as the inability to provide real-time monitoring data. It cannot accurately measure values, requires higher cost, and has low sensitivity. Therefore, our team is looking for a new monitoring method - biosensors to achieve real-time monitoring, accurate detection, and sensitive monitoring.

In order to make our project proceed smoothly, our team visited factories and laboratories to learn relevant knowledge and response methods; we interviewed experts in the field and factory employees to ensure that our solutions were feasible and could truly help Workers who go to factories; go to different places to conduct seminars and popularize science, so that people can understand relevant knowledge, promote our products, and obtain certain feedback and suggestions.

Source: https://mp.weixin.qq.com/s/zhM8p5qLfX4zj0QraMlJEg

After we learned about the dangers to factory employees in the news, we also visited local hospitals and learned that hospitals near chemical plants receive a large number of workers who fall ill due to chemical raw materials every year. Subsequently, we conducted a survey at a local p-cresol factory and asked the factory employees about their joining time and related symptoms. We surveyed a total of 60 employees in the factory, with working experience of less than or equal to one year, less than or equal to five years, and greater than Twenty in each of the five years, and the results were graphically analyzed. As shown in the figure, the employees who had just joined the company (≤1 year) had almost no obvious discomfort. Only one new employee who did not wear goggles as required reported a burning sensation in his eyes, accompanied by tears. However, almost all old employees who have been employed for more than one year have experienced symptoms of physical discomfort. 20% of employees said they had minor lung damage, 40% said they had insomnia and memory loss, and 30% suffered from kidney-related diseases. diseases, and the above three symptoms all show an increase in the number of employees as they work longer. For example, among the lung injuries, two employees had less than or equal to five years of service, and six employees had more than five years of service. The impact on the kidneys is even more disparate, with two employees having less than or equal to five years of service, and as many as ten employees having more than five years of service. Among old employees who have been employed for more than one year, only 10% have no relevant symptoms or no symptoms. It can be seen that even if protective measures are taken, if workers are exposed to p-cresol for a long time, different organs of the body will be affected to varying degrees.

Based on the above investigation results, we realized the seriousness of the situation that p-cresol is harmful to workers, so we asked the relevant person in charge of the factory whether they were aware of this situation and what preventive measures were taken against this phenomenon. Qin, the person in charge of the factory, said that this situation exists in every chemical factory. "Chemical substances will affect people to a greater or lesser extent," the person in charge of the factory suggested, adding that the factory has adopted gas monitoring instruments for monitoring. However, since the boiling point of p-cresol is 202°C, the monitoring instrument can hardly monitor effectively. There are currently online monitoring systems on the market, but the price is high and the company cannot afford the cost. Therefore, it can only provide the most basic protection for employees through some basic protective equipment such as goggles, gas masks, and protective clothing.

In addition, we also visited several other chemical factories in Wuhan about their current testing and protective measures for workers. Among the four factories we interviewed, all factories arranged regular physical examinations for employees and were equipped with the most basic Protective measures are taken to ensure the safety of workers at the most basic level. However, at the higher-end instrument detection level, only two factories have purchased gas detectors, and only one company has adopted a high-end online monitoring system.

When we asked the heads of these four factories: If we design a sensitive and affordable p-cresol biosensor, would the company be willing to buy it? The person in charge said: They are very much looking forward to our output and will recommend it to our sister companies.

After learning about the above situation, we contacted Professor Guo Chengxi, because his research field during his master's and doctoral studies was the impact of phenolic substances on living organisms. We asked Professor Guo how to design a biosensor that can sense p-cresol. Professor Guo introduced to us that previous research found a specific promoter in Pseudomonas, p-Cresol promoter, that can sense p-cresol. , if a reporter gene is added downstream of this promoter, when p-cresol exists in the environment, the promoter will start the transcription and translation of the downstream gene. Through the expression of the reporter gene, we can observe the phenomenon to achieve perception. For the purpose of p-cresol, this technology can be used to effectively monitor p-cresol in the environment to protect the safety of factory workers.

After receiving guidance from Professor Guo, we designed the above gene circuit diagram through group discussion. The circuit diagram includes J23100, B0034RBS, pchR repressor gene, p-cresol sensing promoter, GFP gene and B0015 terminator. In this model Under the condition, pchR continues to express and inhibits the function of the p-Cresol promoter. However, when p-cresol exists in the environment, p-cresol will bind to pchR, the p-Cresol promoter is released, and downstream transcription is initiated, and the GFP protein will be expressed. . When the concentration of p-cresol in the environment is higher, the expression of GFP protein is also higher. Finally, a fluorescence microscope can be used to record the light signal emitted by the sensor.

After the design was completed, we discussed with Calin scholars in related fields, and Calin made a suggestion: the observation cost is higher because fluorescence needs to be observed under a fluorescence microscope. If we could replace it with a color reaction visible to the naked eye, the cost issue could be solved.

5. Second version of p-cresol sensor design

We designed the genetic circuit diagram of the second generation p-cresol biosensor. We replaced the GFP green fluorescent protein with β-galactosidase. We only need to add X-Gal to the environment and the environment can be monitored through color changes. The concentration of p-cresol in . We conducted experimental verification of the second-generation product and passed different concentrations of p-cresol into the second-generation constructed strain. Unfortunately, even if p-cresol was passed in with a very large concentration difference, the color difference in the results was not noticeable.. It is not obvious, so the accuracy of this generation of p-cresol sensor monitoring is very low, and it cannot accurately judge the concentration of p-cresol in the environment. We asked Professor Xiao (Robert), who pointed out that the p-Cresol sensing system derived from Pseudomonas aeruginosa is not suitable for the chassis microorganisms of E. coli, and pchR may be toxic to E. coli. We instead wanted to test p-Cresol using Pseudomonas aeruginosa as the chassis microorganism. Because pchR itself is derived from Pseudomonas aeruginosa, it should be able to adapt.

We asked Professor Xiao (Robert), who pointed out that the p-Cresol sensing system derived from Pseudomonas aeruginosa is not suitable for the chassis microorganisms of E. coli, and pchR may be toxic to E. coli. We instead wanted to test p-Cresol using Pseudomonas aeruginosa as the chassis microorganism. Because pchR itself is derived from Pseudomonas aeruginosa, it should be able to adapt. But Teacher Xiao told us that Pseudomonas aeruginosa is also called "Pseudomonas aeruginosa" in medicine. First isolated from wound pus by Gersard in 1882, it is a Gram-negative, aerobic, long rod-shaped bacterium with only one-way mobility. Pseudomonas aeruginosa is widely distributed in nature and in normal human skin, intestines and respiratory tract, and is one of the more common clinical pathogenic bacteria. It usually affects the lungs and urinary tract, or causes burns, wounds and other blood infections, such as septicaemia. Although very uncommon, Pseudomonas aeruginosa can also cause pneumonia. Moreover, Pseudomonas aeruginosa is a Class 3 microorganism and is not suitable for use in this subject. Therefore, we gave up continuing to use the pCresol sensing promoter component.

After discovering the incompatibility of the p-cresol-sensing promoter, we made major modifications in our thinking. The third-generation gene circuit diagram is shown in the figure above. The first module is the chromogenic module, which mainly performs functions. The component is tyrosinase. Tyrosine is continuously expressed inside the engineered bacteria, and tyrosinase catalyzes its target molecule by involving molecular oxygen in two different reactions, namely the hydroxylation of p-methylphenol to 4-methylcatechol. , 4-methyl catechol is oxidized to p-4-methyl catechol. We use tyrosinase and MBTH. The working principle of this sensor relies on 4-methylo-phthaloquinone, the oxidation product of tyrosinase activity, which is rapidly converted into a pink complex in the presence of MBTH.

The second module is the lysis module. The main functional component is the SRRz cleavage gene. The SRRz gene we used is a linked gene composed of the perforin gene S, the bacteriophage lysozyme (transglycosidase) gene R and the gene RZ. The product of the R gene is a water-soluble transglycosylase, a type of peptidoglycan that breaks down cell walls. The product of the RZ gene is an endopeptidase, which cleaves the oligosaccharides of peptidoglycan

cleaves between and cross-links between peptidoglycan and the outer membrane of the cell wall. The function of the S gene product is to change the permeability of the plasma membrane and form a porous structure on the plasma membrane, allowing the enzymes produced by the R and RZ genes to pass through the plasma membrane and reach the cell wall, thereby acting on the cell wall and breaking the cell wall. , the release of intracellular substances. The functions of both R and RZ gene products are to degrade cell walls. Because bacterial cellulase and algin lyase are expressed within bacteria, but our substrate is outside the bacteria, in order to enable the enzymes we express to contact the substrate and react, we need to lyse the bacteria. In addition, using lytic genes to disrupt cells can simultaneously solve biosafety issues. Therefore, when arabinose is present in the environment, the permeability of the cell membrane of the engineered bacteria changes and the cell wall is degraded, causing the cell contents to be released into the solution. When we culture the engineered bacteria for a period of time and add MBTH and arabinose to the system, a color reaction will occur. The concentration of p-cresol in the environment can be judged by the presence and depth of the color.

We conduct experimental verification of a third-generation p-cresol sensor. Experimental results show that the third-generation p-cresol sensor has considerable sensitivity and can accurately display the corresponding color depth according to the concentration of p-cresol in the added environmental sample. In addition, the cleavage system of the third-generation p-cresol sensor can also function normally to ensure biological safety.

After we completed our laboratory work, we communicated with some workers and hoped that they would use our protocol for testing. The workers told us that our current entire workflow was too complex and difficult to operate for people without biological background. . And most factories do not have standardized laboratories that can complete this content. Therefore, we hope to design a hardware device that can detect the content of p-cresol in the factory. We asked workers about their expectations for this hardware device. Workers reported that they wanted it to be easy to operate, cheap, visual and mobile. We interviewed fifty employees about what they valued most, and found that the vast majority preferred that the p-cresol sensor be easy to operate. “Our cultural level is average, and we will find it difficult if the operation process is too complex,” said the employee.

Based on the above requirements, we designed this device. It mainly consists of a shell, a mobile power supply, an air pump, a time control chip, a reaction bottle, and some supporting reagents. When we gave this equipment to workers for trial use for the first time, there were cases where the air pump was damaged due to misuse by users. Based on this, we added a buffer bottle to prevent damage to the equipment.

We sent the manufactured third-generation p-cresol sensors to the four p-cresol factories included in the above survey. After using them for a week, we conducted a questionnaire survey on these four factories. Out of 5 points, all three companies gave us a score of 4 points. They commented: "This p-cresol sensor is sensitive and easy to operate. It is time to replace the detection system currently in use." Only one company gave it a score of 1 and stated that they only needed to do a good job of protection and did not need such monitoring tools. "We have not equipped monitoring equipment before this, and such expenditure is not within our plan." The person in charge of the company replied. Although we have received such feedback, we will still focus on the problem, work hard to optimize the design, reduce costs, and make the product price more acceptable.

After the production process of the third version of the p-cresol biosensor hardware matured, we went to the land and water sources downstream of the p-cresol factory to monitor p-cresol. After monitoring, we found that the downstream water sources were seriously polluted by p-cresol. In contrast, land pollution is lower. We asked the relevant person in charge of the factory. The person in charge said that the traditional monitoring instruments used before were not sensitive enough. As a result, even if the factory carried out sewage treatment, it could not accurately monitor whether the treated sewage met the standards. The discharge of substandard sewage caused downstream problems. Land and water sources have been polluted to varying degrees.

After a series of experiments and practices, we are confident that we have obtained a sensitive, low-cost, and accurate p-cresol biosensor. So we went to Wuhan Middle School to conduct science popularization activities and held seminars for high school students. The common purpose of these activities was to publicize the dangers of p-cresol to the public, remind everyone to take relevant protective measures, and let more people know about it. Next, we will introduce to you the p-cresol biosensor we designed, which can effectively solve the monitoring problem at this stage. However, monitoring can only serve as a warning. How to design a new type of sensor in monitoring In addition, it can also degrade p-cresol at high concentrations, which needs to be studied by future teams.