Team:HZAU-China/Safety
This year, we have done a lot of work in order to meet the requirement of biosafety. After collecting opinions from experts and the public, we learned that the general concerns are about: How is it safe to apply bacteriotherapy against cancer without causing unexpected damage or infection? We took these concerns seriously and have found some practical solutions.
Salmonella enterica serovar Typhimurium str. SL1344 is not entirely harmless. In fact, the strain SL1344 which we chose for our project may as well cause slight enteritis. However, we chose it base on the fact that it is a well-learned intracellular pathogen with a lot of engineering experiences we can refer to, which is suitable for delivering GSDMD into cells. So we finished the Check in Form and the Safety and Security Form carefully to make sure the information is clear and intact as well as we can.
Gene sifA is a vital gene located in Salmonella pathogenicity island, which increases the stability of Salmonella-Containing Vacuole (SCV) after infection. We knocked out the sifA gene from Salmonella Typhimurium str. SL1344 genome by conjugation transfer. The aim is to reduce the toxicity of Salmonella itself, then to allow the GSDMD protein, which is able to rupture the phospholipid bilayer structure, to be released into the cytoplasm of a tumor cell. After gene knock out, the strain can be easily controlled by human macrophage. See also Design and Results pages for details.
Salmonella has natural taxis towards tumor microenvironment. Base on the results of public survey and some opinions from experts, we decided to enhance the targeting specificity to a new level. We designed a new circuit to introduce OmpA-RGD protein as a surface recognition molecule, where RGD is a well-studied tumor homing tripeptide that binds specifically to alpha v beta 3 (αvβ3) integrin, a protein widely distributed on the surface of tumor cells. We successfully displayed RGD on the surface of a bacteria strain, and also proved its targeting ability towards αvβ3. See also Design and Results pages.
In our lab there is a safety training for everyone which covered the following aspects:
1. Personal peripheral wear: Lab coat and gloves must be worn all the time during work. Personnel must wash their hands after experiments, or before they leave the laboratory working areas. Open-toed footwear must not be worn in laboratories. Eating, drinking, smoking, applying cosmetics, handling contact lenses and sleeping are prohibited in the laboratory working areas.
2. Chemical safety: We printed "Dos and don'ts when using hazardous chemicals" and posted them on marked place. Students are trained to use dry powder extinguishers and carbon dioxide fire extinguishers in our lab.
3. GMO safety: No biological materials are allowed to be brought out of our lab. Strains must be put into death before dumping.
4. Study of guidelines: We listed some biosafety websites used for team member education:
Figure 1. Lab safety seminar
Govern biosafety in research laboratories:
http://english.biosafety.gov.cn/
Biosafety guidelines of our institution:
http://swx.hzau.edu.cn/
Regulations that govern biosafety in research laboratories:
http://english.biosafety.gov.cn/
Our lab locates in the building for School of Life Science and Technology, Huazhong Agricultural University. An access controlling system prevents unauthorized person from entering, and one has to show the student ID card to get into the door. Our biological wastes are managed by trained stuff, therefore will not enter the water body or contaminate the outside environment.
Figure 2. Door access control
Here we illustrate that our 2018 project did not involve human experimentation, gene drive, use of animals, or use of any parts from risk group 4 organisms. Our compliance with the “Do Not Release”, “Deletions as Modification” and “Anti-Microbial Resistance (AMR)” policies is demonstrated above, as we always take care of what we use and never let experimental organisms with resistance plasmid or genome modification out of the lab alive without handling. As for the Human Subject Research policy, our survey is basically about the public’s opinions or attitude towards newly emerged cancer treatments, which barely involved any ethical issues. Nevertheless, we referred to the “Guidelines on Ethical Review of Biomedical Research Involving Human Subjects (NHFPC, 2016)” and the “International Guidelines on Market Views, Social Surveys and Data Analysis (ICC/ESOMAR, 2016)” before designing the questionnaire, for the protection of the public. All the safety rules are also obeyed; see previous descriptions.
1. Understanding Biosecurity; National research council of The National Academics, Rita r. Colwell
2. Laboratory biosecurity guidance; World Health Organization, September 2006, WHO/CDS/EPR/2006.6
3. Laboratory biosafety manual, Third edition; World Health Organization, Geneva, 2004
