Safety

Introduction

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 form.

Lab Safety

All laboratories of universities in China must be accredited by the Office of the Ministry of Environment following the standard. This year, the 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, follow laboratory hygiene rules and proper disposal of hazardous substances. Also, experiments were first presented in detail by our two supervisors to eliminate biosafety concerns.

Microorganisms review

Protein expression for diagnostic tests was performed in non-pathogenic Escherichia coli strains (E.coli Nissle 1917), Lactococcus lactis NZ9000 and yeast (EBY100). These gene carriers are probiotic or at least harmless. They do not possess serious illnesses or lead to other problems for people or the environment. In addition, they are on the whitelist and belong to BSL-1 organisms.

Experiments

The engineered probiotics were designed to express 20ipaD nanobody on their surface by transforming recombinant plasmids with optimized genes. Thus, they can combine with the antigen on the surface of Shigella. Since Shigella is one of the BSL-2 organisms, we decide to express our targeted antigen in probiotics that we used to express the antibody. We optimized the DNA sequences which code the antigen by using machine-learning software. Using yeast as an example, amino acids are essential for the growth of yeast. Amino acids are absorbed by human bodies in the small intestine. There is a competitive absorption of amino acids between intestinal epithelial cells and yeast. This prevents the overgrowth of yeast cells, preventing the destruction of bacterial flora structure. Using yeast as gene carriers limits the leak of antibiotic-resistant genes, compared with using bacteria as carriers. In addition, galactose was used to induce the expression of the antibody and antigen.

Since we used antibiotics resistance genes, we also pay attention to the potential risks of the leak of antibiotics resistance genes during wetlab process. Discarded liquids and plates that contain antibiotics were placed in specific vessels and then autoclaved.

Harmful reagents and procedures

Part of the planned experiments had to be done by using harmful reagents or procedures. All team members follow the rules under the guideline 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 for visualizing agarose gel, combined with standard laboratory procedures with blue light transilluminator.

Antibiotics were used to select transformants. They can be harmful to sensitive groups. Situations of allergies 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.

Covid-19 associated safety measures

Team members followed all the government and university guidelines based on the pandemic situation of Covid-19 in China. For April and May, team meetings were online because of school closure and in-person activities were allowed after deblocking. Covid-19 tests were performed regularly and all team members got vaccinated.

When wet labs began, team members' attendance was recorded. The usage of lab equipment was registered. All working surfaces were cleaned with disinfectant fluid before and after conducting wet labs.