Safety has always been an important issue we paid attention to, including the safety of project design, lab safety and so on. There is no doubt that both our team members and the feasibility of our project have benefited from the safety rules and training. Which means our team had taken many measures to guarantee the safety of our project, such as setting strict rules regarding lab safety, concerning the potential problems about the design of project, and so on. We think it is our duty to seriously consider safety issues from the beginning to the end.
Project design safety
The aim of our project is trying to construct a light-controlled genetic engineering machine for degrading ARGs by the technology of CRISPER/Cas9 and a module regulated by Inducible promoter for cell lysis. Considering the biosafety of our experiments, we have adopted the measures as follow:
For the safety of our experiments ,we select E.coli BL21,DH5α,E.coli MG1655 as our host organism in our project. These chassis microorganisms are commonly utilized in laboratories and have not possessed any characteristics that can endanger the human body and the security of environment. Thus, our project is safe from the perspective of strains we used.
The plasmids we use in our project (pGLO-Cas9, pTargetF-p15A, dusk-Cas9-pUC57, dusk-eGFP-pUC57, pGLO-lacI, pGLO-cI,pGLO-lysis for E. coli) are almost harmless for humans. Consequently，plasmids lead to no safety issues.
Our parts we utilize in our design all meet the safety requirements .During the time of designing, we ensure that none of the parts assembled into our genetic engineering machine is able to take a toll on health in existent researching files. As a result, we confirm that all parts wouldn’t act as virulence factors.
4.Secure genetic circuits
In order to further improve the security of project, a module for cell lysis is constructed to disrupt the cell after eliminating the ARG. We use lysis gene as the functional element, the cells wall will be split once the inducible promoters are induced .Besides, in our project ,we choose the GFP and eGFP as reporter genes which can be detected directly without adding any harmful reagents.
Firstly，all team members have been trained about lab safety by a professional researcher. And only passing a biological safety test can we enter the laboratory. Then, in order to prevent our members from encountering dangers in the process of experiments, we make many safety requirements for the laboratory .This includes, but is not limited to, the following regulations:
●To enter the laboratory, you have to wear lab coats
●Smoking, eating, and speaking loudly are not allowed in the laboratory.
●The laboratory should be kept clean in any time. Special chemical or biological waste should be discarded into corresponding waste bin.
●The glassware in the laboratory should be cleaned right after it is used.
●If waste is microbial contaminated, it should be sterilized to ensure its safety.
●When high-pressure, heating, or sterilizing equipment is being used, there must be someone be left in the lab to look after the equipment to ensure safety.
●Bringing unrelated personnel into the laboratory is strictly prohibited.
●After using the equipment, power supply should be cut in time. Check the doors, windows, power supplies, equipment in aseptic lab, and so on before leaving the lab, especially before holidays.
●Any manipulations requesting for sterile environment should be performed in a bio-safety cabinet.
●Record the experimental process and the experimental results in time.
For the sake of shipment safety, we have followed the iGEM official advice for Chinese Teams to send our parts to GenScript in Nanjin, China .Hence, the DNA parts can be guaranteed the safety.