Describe all the safety issues of your project.
The Duke Kunshan University (DKU) iGEM team sets safety as an integral part of science. It includes protection for sensitive groups and the environment. For this reason, safety was considered from two perspectives—inside and outside the laboratory. For more detailed information, please check our safety and check-in forms.
All laboratories of universities in China must be accredited by the Office of the Ministry of Environment following the standards. This year, the whole project was conducted in the BSL-1 laboratory. Team members wore lab coats and gloves during experiments. Experiments related to bacteria were performed in the biosafety cabinet and near butane burners.
Before the laboratory started, the team received safety training including how to use equipment properly, laboratory hygiene rules, and properly dispose of hazardous substances. Also, experiments were first presented in detail by our two supervisors to eliminate biosafety concerns. Under the guidance of our two supervisors, weekly lab equipment checking was conducted.
Plasmid amplification and protein expression were performed in non-pathogenic Escherichia coli strains (E.coli DH5α, E.coli BL21). These gene carriers are harmless. They do not possess serious illnesses or lead to other problems for people or the environment. In addition, they are in the whitelist, belonging to BSL-1 organisms. We also cared about the pathogen we targeted. The alternative to our plant pathogen Candidatus Liberibacter asiaticus (CLas) is Liberibacter crescens (Lcr). Lcr is a model organism commonly used in studying Citrus Greening (Huanglongbing) [1]. Considering it is not on the whitelist, our team sent a check-in form to the iGEM committee. Besides, Lcr belongs to Risk Group 1 based on the classification of the iGEM committee because it normally does not cause harm to human bodies. To test the efficiency of RNAi, we used the entomophila of Citrus Greening. After receiving the reply from the iGEM committee that we did not need to send an animal use form, we only sent the D. citri check-in form. The bugs were kept within nets and did not process serious illnesses to humans.
Since we used antibiotics resistance genes as our selection genes, we also paid attention to the potential risks of the leak of antibiotics resistance genes during wet-lab process. Discarded liquids and plates that contain antibiotics were placed in specific vessels and then autoclaved. For engineered microorganisms, we autoclaved them before disposing. To avoid spreading our products to the outside environment, we only did experiments within labs.
Part of the planned experiments had to be done by using harmful reagents or procedures. All team members follow the rules under the guidelines provided by our lab manager and assistants during experiment safety training.
UV light was used to sterilize the biosafety cabinets and equipment inside the cabinets. Safety measures were taken to avoid people's direct exposure to UV light. The gel imaging system was used to visualize agarose gel, combined with standard laboratory procedures with a blue light transilluminator.
Antibiotics were used to select transformants. They can be harmful to sensitive groups. Situations of allergy of team members were collected. Antibiotics were well stored and Handled with proper safety measures according to disposal protocols.
Other hazardous chemicals, such as Tris-HCl and methyl alcohol were stored and relevant procedures were conducted in chemical ventilation cabinets. Liquid waste was handled with appropriate safety measures.