Team:YAU-China/Safety
Safety Overview
The safety of biological experiments is a major problem throughout the project. We take safety seriously! We believe it is of vital importance in iGEM competition, and even more important for us. The track we chose this year is Therapeutic Track. Therefore, safety has always been a serious issue we considered from the design of the project all the way to lab work. Our project provides a new way to solve the problem of burn infection caused by biofilm produced by Pseudomonas aeruginosa. We have set strict rules regarding lab safety to ensure the safety of using Pseudomonas aeruginosa. In addition, we also designed a variety of safety measures to prevent the experimental strain gene from escaping.
Lab Safety
We carefully assessed the risk of our project, including the organism we used, experiment conditions, training and waste management. All information is summarized in our safety form. Specifically as follows:
In order to make our experiments as safe as possible,before we were allowed to enter the laboratory, Dr. Liu, our experimental safety officer, strictly trained us in biosafety education and project safety design.He informed us of all the potential risks and dangers associated with the usage of different chemicals, techniques, and equipment, and how we could minimise these risks. Following this, we were given a laboratory induction by our instructor, who demonstrated the safest and most efficient ways to carry out the protocols we would require.
We further reported our situation to the safety committee with a check-in form, and was later granted permission to use Pseudomonas aeruginosa in our project. We used Pseudomonas aeruginosa PAO1 as chassis bacteria to construct attenuated Pseudomonas aeruginosa strains by knocking out the genes related to exopolysaccharide synthesis of Pel、Psl and type III secretory system(T3SS) structural protein. We also used E.coli , TG1, S17-1, Transetta (DE3) and TransB (DE3) for gene cloning, parental hybridization and protein expression. Our laboratory is BSL-2 laboratory, which can meet the use conditions of Pseudomonas aeruginosa wild strain PAO1. The second-level biosafety cabinet in the laboratory is used for the related experiments of Pseudomonas aeruginosa . To protect lab personnel from infection and other hazards, our team members always wore laboratory gowns, gloves and masks during operation. We modified the wild Pseudomonas aeruginosa to prevent the potential danger of engineering bacteria leakage. Our team members have received training before conducting the experiments under the supervision of trained personnels (lab technicians and senior graduate students). We modified the wild Pseudomonas aeruginosa to prevent the potential danger of engineering bacteria leakage.
To avoid gene modified organisms being released into the environment, all materials, including used medium, agar plates, pipette tips, will be sterilized before disposed. To avoid potential contamination to the environment by Pseudomonas aeruginosa ,we designed a variety of security mechanisms and application models in our experiments. In order to further ensure the safety of our experiment, we went to Yan'an People's Hospital to have an exchange with Vice-Director of Burn Junjie Ding. Vice-Director Ding affirmed our project very much. (This is also a part of our human practices.)
Project Safety
Pseudomonas aeruginosa is one of ubiquitous gram-negative rods which we can see everywhere. It is also called hospital-acquired infections because it has multidrug-resistance which ensures its survival state. Also, Pseudomonas aeruginosa is an opportunistic extracellular pathogen to human. Because this bacterium is responsible for some diseases such as acute infections with burn wound and its primary hazard is the infections during surgery.
Pseudomonas aeruginosa T3SS is a very important virulence factor in acute human infection. To ensure the safety of our engineered bacteria, we knocked out the T3SS gene and made it a attenuated strain. Then PrtN was overexpressed and lysozyme was expressed in large quantities to induce the lysis of engineered bacteria and ensure our engineering bacteria will not pollute the environment as well as human body. In addition, we knocked out two genes, pelF and pslA-B , which are crucial for biofilm synthesis, so that our engineering bacteria do not produce biofilm. The fabV gene of oleoyl ACP reductase was added to the plasmid as a biological screening marker to solve the problem caused by plasmid loss. Finally, our team designed a band-aid model to prevent engineering bacteria from escaping.
General Safety
Our microbial laboratory level is BSL-2, which can meet the operation requirements of Pseudomonas aeruginosa PAO1.
There is no accumulation of debris around the laboratory. Arrange daily duty to ensure the safety and cleanliness of the laboratory.
Our iGEM lab is equipped with safety apparatus such as fire extinguishers, blankets and first aid kits in case of dire situations.
The equipment used in the laboratory is tested by the relevant departments. All toxic reagents are kept in isolation and used in isolation, such as ethyl acetate, trichloromethane, etc. We will put them in the labeled storage cabinet and use them in specific areas.
