In our project, the bacteria strains we only used are E.coli DH5α, DH10B and BL21(DE3) that are not harmful to human bodies. All relevant experiments were performed on a clean bench to avoid contamination of bacteria. Furthermore, we experimented with all bacteria strains in a laboratory and followed laboratory waste disposal rules. Discarded engineered strains will be sterilized at 121℃ for 20 min before being discarded as medical waste so that our engineered bacteria do not spread in the natural environment. In addition, when the engineered bacterial strains are subjected to heat-induced lysis, we seal the bacterial cells in a clean bench, and then conduct the measurements of lysis process in a microplate reader.
Besides using 4T1 cells to conduct CCK-8 experiments, the relevant experiments were arranged in a BSL-2 lab in Shanghai University of Chinese Medicine to ensure the safety requirements. All the relevant experiments were conducted under the supervision of our instructor and the cellular chamber only allowed the trained to get inside for special operation.
Our DNA materials were directly ordered from Genscript and no hazardous chemicals were used in this project except for one toxic chemical reagent, the nucleic acid dye, to prevent nucleic acid contamination. We isolated the containers from which they came into contact and used physical isolation to protect the experimenters themselves. In addition, We sealed all our mediums to avoid contamination of the bacteria as well as surroundings.
One of the living organisms involved in this research are mouse breast cancer 4T1 cells, which we have submitted in the satefy form. During the experiment, we followed bioethics and the laws and regulations of the People's Republic of China and other countries and complied with them. Below are the sources of the rules, regulations and ethics we adhere to.
1. https://iep.utm.edu/bioethics/
2. https://www.mee.gov.cn/ywgz/fgbz/fl/202303/t20230314_1019536.shtml
2. https://www.fda.gov/drugs/cancer-drugs/cancer-drug-approval-endpoints
Before starting our experiments, we were required to take the safety training sessions and several laboratory rules:
1. When we enter and leave the laboratory, we must wear laboratory clothes, masks and gloves. Do not wear lab coats and gloves into the living area except when we need cargo elevator transportation.
2. The experiments should be carried out in the designated laboratory.
3. Eating and drinking is strictly prohibited in the laboratory.
4. Do not carry the bacteria to any other areas to avoid exposing infectious specimens to.
5. If using public instruments, we must be accompanied and guided by the instructor.
6. The use of laboratory instruments should be trained and approved by the instructor. Special instruments (such as autoclaves) need to be used under the supervision of qualified personnel.
7. In experiments, the clean bench was used to provide a sterilized environment for some experiments that involve bacteria and cells.
8. When we did cellular experiments, we must not place our hands above the medium to prevent cells from pollution.
9. In the BSL-2 laboratory, we need to wear isolation slippers, and other organisms cannot be brought in. The equipment in the BSL-2 laboratory cannot be used for other purposes.
10. Chemical reagents would be discarded into the liquid waste container, contaminating reagents need to be isolated.
How to use centrifuge
1. Place the solution to be centrifuged into a properly sized test tube and place it in a centrifuge using a central symmetry method.
2. Pay attention to the centrifugation time, some do not need to be centrifuged for too long.
How to use the clean bench.
1. Before use, turn off the UV light, turn on the exhaust fan and exhaust.
2. All items must be disinfected before entering the operating platform.
3. After using the operating platform, it is necessary to confirm that there are no bacteria or other non disinfectable items inside, and then turn on the UV light for disinfection.
How to use pipette gun
1. Before use, check the measuring range and select a suitable pipette gun.
2. Choose the appropriate gun head.
3. When taking liquid, extend the gun head below the liquid level, press the button until resistance is felt, that is, slowly release it after the first gear to ensure that enough liquid is sucked in.
4. When discharging liquid, extend the gun head below the liquid level, press the button to the bottom, which is the second gear, and press and hold the button to prevent the liquid from being sucked back, causing the gun head to leave the test tube.
5. The used gun head must be removed and replaced before continuing to use.
6. The pipette is a precision instrument that must be handled with care.