Team:SDSZ China/Safe
Team:SDSZ China
Safety is always the most important issue in every experiment. While we were proceeding with our experiments, we took safety as the first thing on list to consider in our design. We considered safety in several different aspects: safety in the lab, safety during operation, and biosafety throughout the whole process. In order to ensure the safety issue in every step, we did detailed investigation on the Internet and made sure that we understand the principles of biosafety and biosecurity.
Preparations
We did all of our experiments in The Lab of Insect Ethology and Pest Control in China Agricultural University, an advanced molecular biology lab in the building of crop protection in CAU. We made sure that we do not use any toxic chemicals that may harm the environment after leakage to conduct our experiment.
Safety during Experiment
While working on the synthesis of enzyme luciferase to catalyze the oxidation of D-luciferin, we used a few chemicals. While running electrophoresis, we avoided anything that can raise safety risks. Instead, we used Gelstain and GelRed to dye agarose gel, which is way more safe than EB. We also adopted E. coli (strainBL21/DE3 and Dh5a). The cells are not able to propagate outside the lab without suitable growing environment. Thus, even if leakage occurs, the environment will not be contaminated. In addition, all the substrates of our reaction are totally safe, including D-luciferin, magnesium ions and tea polyphenol.
We were under strict trainings before being allowed to enter the lab and conduct experiments. The PI of the lab, Xuewei Yin, offered every team member a thorough instruction for a complete week to help with the operations, including electrophoresis, spectrophotometer, and PCR. Students need to pass a test before entering the lab. When conducting experiments that involve dangerous equipments, such as cutting agarose gel under ultraviolet light for gel extraction, we are required to put on necessary gloves and goggles. What’s more, eating or drinking is strictly prohibited in the lab, which ensures a clean and safe environment in the lab.
Protocol
We conducted the experiments under the supervision of at least one other teammate, and the wastes were collected collectively and followed the university’s biology department's waste treatment procedures strictly.
There are lots of safety issues regarding the experiment equipments. Therefore, we undergone strict trainings and practiced for long before started our experiments. For instance, when we were doing experiments in the sterile operation area, we were not allowed to open the device when the ultraviolet light stayed on for at least 20 minutes. When we are used the burner to complete sterile operations, we made sure to operate with the flame in a proper distance, at which the environment is sterile and the flame would not hurt us. When we were using the autoclave, we were asked to follow the instructions strictly so that we need to wait until the warning light turn off and the pressure is released, since it is dangerous for the laboratory technician to continue operation in such a high pressure and high temperature status. After finishing those trainings and practices, we were given an exam to test our operation skills. During the test, we need to know how to deal with emergency. For instance, if we happen to turn down a burner, we need to cover the flame with wet towel immediately, and do further managements. The tests were scored and everyone were able to be familiar with the correct operation of the equipments.
The safety issues of reagents were also taken notice of. For instance, TEMED, which has strong neurotoxicity, serves as a coagulant when preparing SDS-PAGE gel. To avoid the harm of TEMED during the experiments, we were asked to finish making SDS-PAGE gel in the fuming cupboard, where air system can expel the volatilized TEMED. We were also asked to seal the bottle of TEMED as soon as we get it. The GelStain, which is slightly radioactive and toxic, is used as dye in electrophoresis. When we were adding GelStain into the gel, we wore special gloves, which are only used in this specific step to finish the operation.
The culture solution, which was used to culture bacteria for protein translation, need to be dealt after we finish the experiment. We collected the culture solution and stored it in a jar. We inactivated the waste culture solution in a high pressure and temperature condition. After those operations, we store the solution in a sealed jar to be discarded with standard protocols by the lab and biological companies in charge.
Potential Risks and Prevention
We were only exposed to organisms with the lowest risk group, and we handled mostly nonhazardous chemicals like agarose, LB, MSB and SOB medium. But we also used the neurotoxic chemical polyacrylamide to run the polyacrylamide gel electrophoresis. In order to prevent any potential risks, we wore gloves and lab coats in lab, and we performed all operations in a bio safety cabinet when handling toxic chemicals.
While we dealt with coliphages for detection in the latter part of our experiment, we made sure that all operations were conducted on clean bench. After the sample was taken a picture of and analyzed, we turned on ultraviolet light for 30 minutes to kill the phages remaining in the bench. Followed by the radiation was the boiling of the samples. Since phages are vulnerable to ultraviolet radiation and hot temperatures, these steps can successfully prevent phage contamination to the environment.
Our secondary PI is doctor in genetic engineering and oversaw the most part of the experiment. The lab PI and other graduate students working in the lab were responsible for the safety and security issues of the lab. For the biosafety officers, they evaluate our experiment design and point out any safety issues in our design. If there are any safety issues, they help us modify our design. Before starting our experiment, they gave us instructions and lots of training to help us finish the operation safely. During the experiment, they stayed in the office next to the lab and gave us help as soon as we encounter any problems. Since they have abundant experience in genetic experiments, they could deal with problems effectively.