1. Project Safety

In our first and second-generation para-cresol sensors, our primary focus was on the colorimetric module, as it directly reflected our monitoring results. However, we overlooked the issue of biosafety.

Later, we introduced a second module, the lysis module, with the key component being the SRRz lysis gene. The SRRz gene we used is composed of a concatenated sequence of the puncturing element gene S, the phage lysozyme (β-glycosidase) gene R, and the gene RZ. The product of the R gene is a water-soluble β-glycosylation enzyme that degrades the cell wall's peptide polysaccharides. The product of the RZ gene is an endopeptidase that cleaves between the oligosaccharides of the peptide polysaccharides and cross-links between the peptide polysaccharides and the cell wall's outer membrane. The role of the S gene product is to alter the permeability of the cell membrane, creating a porous structure on the membrane. This enables the enzymes produced by R and RZ genes to pass through the membrane, reach the cell wall, and cause cell wall disruption, leading to the release of intracellular substances.

Both the R and RZ gene products function in degrading the cell wall. As cellulase and alginate lyase are expressed within the bacteria, but our substrate is located outside the bacterial cells, cell lysis is necessary to ensure that our expressed enzymes can come into contact with the substrate and react. Additionally, cell lysis addresses the issue of biosafety. Thus, when arabitol is present in the environment, changes in the bacterial cell membrane's permeability and cell wall degradation occur. This results in the release of cellular contents into the solution. After culturing the engineered bacteria for some time and introducing MBTH and arabitol into the system, a color reaction occurs. The presence and intensity of the color are used to determine the concentration of para-cresol in the environment.

We conducted experiments to validate our third-generation para-cresol sensor. The results demonstrate that the third-generation para-cresol sensor exhibits remarkable sensitivity and accurately reflects the concentration of para-cresol in the environmental samples added. Furthermore, the lysis system of the third-generation para-cresol sensor operates effectively to ensure biosafety.

2. Laboratory safety

Before entering the laboratory, we took a course on "matters needing attention in laboratory safety", which mainly included the following points:

(1) when operating in the ultra-clean worktable, do not operate under ultraviolet conditions to avoid burns. After spraying and sterilizing with ethanol, wait for the ethanol to evaporate before igniting in case of fire. In addition, alcohol lamp alcohol is not too full, 1/2 to 2/3 will be fine. In the process of alcohol lamp burning, do not leave without authorization, be sure to turn off the alcohol lamp before leaving, otherwise it is prone to fire.

(2) the operation of volatile chemicals should be carried out in an open ventilated kitchen and wear masks and gloves at the same time.

(3) when opening the sterilization pot, wait until the temperature drops below 60 degrees to avoid steam scald.

(4) when using the centrifuge, it must be balanced and placed equally, and check whether the built-in gasket is installed, and cover the inner cover and outer cover of the centrifuge before it can start operation.

(5) for toxic reagents, multiple protection should be taken during operation. For example, when using EB, wear double gloves and throw away the outer gloves in time when you get out of the glue running room, so as not to cause secondary pollution and affect the health of yourself and others.

(6) the broken glass should be cleaned in time, and the broken or cracked glassware should be discarded into the cardboard box in figure 1 immediately and shall not be used any more. Cutters, carving knives and scissors should be put away and put away after use to avoid cutting others. Needles, toothpicks and other wastes should be trapped and thrown into the sharp box in the sink in the middle of the laboratory (figure 2) to avoid harming garbage disposal personnel.

(7) High-power appliances (including microwave ovens, induction ovens, ovens, ventilator) that are not used at night should be unplugged when leaving.

Before entering the laboratory, we also signed the "letter of responsibility for Laboratory Safety" in accordance with the regulations, which read:

1. When students enter the laboratory to carry out experiments, they must abide by the rules and regulations and operating rules of the laboratory.

2, before the experiment, students must carefully preview the experimental teaching materials, make clear the purpose, steps and principles of the experiment, and answer the questions of the instructor before they can carry out the experiment. Students must observe the experimental time, do not be late, leave early, and do not be absent from work without reason.

3, enter the laboratory to dress neatly, it is strictly forbidden to wear slippers and vests. The room should be kept quiet and clean during the experiment.

4, after the experiment is ready, it must be checked by the instructor before the experiment can be carried out; strictly according to the requirements of the experiment, correct operation, careful observation, in-depth analysis and thinking, and truthful records; and complete the experiment on time with quality and quantity.

5，Take good care of instruments and equipment, save water, electricity and consumables, do not use instruments, equipment and other articles that have nothing to do with this experiment without permission, and do not take any items out of the laboratory outside.

6. Pay attention to safety during the experiment. If an accident occurs, you should immediately cut off the power supply, protect the site, and report to the instructor in time.

7. After the completion of the experiment, the power supply, water and gas should be cut off in time, and the instruments and equipment used should be restored to their original position. Do not leave the laboratory until the instructor inspects the instruments, tools, materials and experimental records.

After the experiment, the experimental report should be carefully written according to the requirements, the experimental results should be carefully analyzed, the experimental data should be processed accurately, and the original data should not be changed.

9. when students want to use the open laboratory to do self-designed experiments, they should contact the relevant laboratories in advance to report their own experimental purposes, contents and required experimental instruments and materials, and with the consent of the teacher in charge, carry out the experiment within the arranged time.

Parents are not allowed to follow students into the laboratory.

11. Whoever violates the above-mentioned provisions shall be dealt with in accordance with the relevant provisions according to the seriousness of the circumstances and the degree of understanding.

I promise to seriously study the "Laboratory Safety Management measures (trial)", and during the experiment, strictly abide by the rules and regulations in the documents, obey the management of the laboratory teachers, ensure the safety of the laboratory and ensure their own safety; use instruments according to correct operating procedures. If a safety accident occurs because of my violation of normal operating procedures, resulting in personal injury and property loss, I am willing to bear the corresponding responsibility.