Team:SCUT China/Safety
Our in biological experiments is always important in biological studies. To keep laboratory activities safe is not only responsible for the project itself, but for the society and the nature. Therefore, we are obligated to formulate policies and procedures of risk assessment. Also, to implement basic principles of research and development as well as production safety, our team has carried out the safety risk assessment from July 15 to July 22 in 2019.
Lab Safety
1. Our laboratory
The laboratory is equipped with enough work and rest areas, storage facilities that meet the demands of preservation and protection, an air conditioning system which ensures laboratory working temperature and good ventilation, adequate lighting, and a fire alarm system that can work normally. In addition, all kinds of facilities in the laboratory will be maintained regularly.
2. Access to the laboratory
We did several seminars directed by Professor Yang Xiaofeng, Dr. Lu Yanping and the graduate students to teach the basics of the laboratory. Moreover, as a security measure, only biotechnologists and biomedical engineers could be there without surveillance due to the experience and knowledge acquired during the degree. Over the course of these seminars we were informed about the evacuation protocols, waste management and our rights and liabilities as SCUT_China team members.
3. Waste management
Project team members can consciously clean the wastes and put them into special experimental dustbins. Discarded medium and sharp tools will be placed in specific bins and handled by professional institutions. The experimental wastes will be sealed and sterilized with high-pressure steam before being discarded to ensure that microorganisms in the laboratory will not be leaked to the environment.
Project safety
1. Strains
We verified the feasibility of database building by adjusting the acid-fast factor of our strains, which requires us to genetically modify the bacteria. To minimize risks, we used the non-pathogenic chassis Escherichia coli DH5α and Bacillus subtilis 168 to produce our proof-of-concept model and designed our product to be solely used in laboratory settings. Escherichia coli DH5α and Bacillus subtilis belong to risk group 1, which allowed us to keep our experiments in ML-1 laboratories.
The T7 RNA polymerase（RNAP） and its native promoter LacUV5 promoter were incorporated into the genome of E. coli K12 MG1655 by using the CRISPR-Cas9 system. In our tests, the T7 promoter can work in E. coli K12 MG1655-T7 RNAP. E. coli K12 MG1655 belong to risk group 1, which allowed us to keep our experiments in ML-1 laboratories. We transformed pCas into E. coli K12 MG1655. Cas9 was induced by arabinose and would not be expressed under normal circumstances, making E. coli K12 MG1655-Cas9 more stable. CRISPR guide RNA target poxB from the genome of a prokaryote, not human genes which meet iGEM safety policies.
3. Acid resistant strain
We make use of VerProS pool to select the best acid-resistant strain. E.coli K12 MG1655 belong to risk group 1, which allowed us to keep our experiments in ML-1 laboratories. We will strictly follow laboratory safety procedures during the operation, and all waste strains and media will be sterilized by autoclaving. We will strictly follow the “Do Not Release Policy”. We will not release or deploy any genetically modified organisms, or the products of genetically modified organisms, outside the lab.
4.Working part plasmid
In order to regulate acid resistance of engineered bacteria，we construct our working part plasmid with gene ybaS, gadC, gadB, katA and some synthetical short fragments by the supplier. The genes will be cloned from the genomes of E. coli K12 MG1655 and Bacillus subtilis 168. And we also apply the Golden Gate Technology to DNA assembling, which is same for the way of VerProS pool except for using CmR antibiotic. In summary, all the engineering bacteria are Bacillus subtilis 168 and E.coli from strains DB3.1, DH5, MG1655, which are classified as Risk 1 so the harm can cause to people is minimal.
To construct the Promoter-Toehold Switches pool -- VeProS pool, we apply the Golden Gate Technology to DNA assembling, as it is a method that allows the highly efficient directional assembly of multiple DNA fragments perfecting for our pool of 10^4 combinations. The negative selection of non-transformed bacteria will be performed by using Kan antibiotic whereas the positive selection of the transformed bacteria with the recombinant plasmid will be carried out including ccdB lethal gene in the destination plasmids. The CcdB protein is lethal to most of the BioBrick cell strains, only E.coli DB3.1 is resistant. Consequently, we use DB3.1 as chassis to construct pET30-ccdB, DH5α to construct the pool, and MG1655 as the last destination chassis. All the engineering bacteria are E.coli from strains DB3.1, DH5, MG1655, which are classified as Risk 1 so the harm can cause to people is minimal.
E. Coli DH5α is used for isolation of vector pUC19 and pET-30(a) as well as plasmid amplification, which is an organism of Risk Group 1 and also in the white list. There are no risks of infections for the strain does not have any pathogenicity. Restrict digest and ligation as well as Golden Gate are applied in plasmid constructions, with all reagents merely working on bacterial genome. Additionally, no gene editing technology is involved in our modifications. Carbenicillin and kanamycin are used to screen the requiring transformants, which can effectively inhibit microbes without resistance and no bacterial strains in our laboratory show drug resistance. All safety and security rules are followed during the whole experiments.
Risk Assessment