In biotechnologies, microorganisms are widely used for a variety of purposes, to explore new ideas and new possibilities to improve our everyday life. In this context our DAISY project aims to develop a new way to detect allergies by using genetically modified organisms. Our lab has an authorization to handle GMOs of class 1. During our manipulations, new GMOs were created through molecular biology processes, and we cannot totally rule out any putative impact outside of the lab even though the origin of genes were related. We had to assess our lab product that may contain GMOs. In adequation with the Nagoya protocol, we will explain here what principle we have followed during our wet lab, as well as during the design process to respect legislation and the environment.

Microorganisms classification

The detection process of IgE proposed by the DAISY project is based on only one bacterial species, Escherichia coli. In addition to its multi-purpose use, we have chosen E. coli for its harmlessness and its good global knowledge in the literature to ensure a high safety of manipulations. It is compatible with the level 1 laboratory used for this project. In that way, E. coli (DE3) Tuner, E.coli Stellar and E. coli DH5α were used, all of them belonging to the white list set up by iGEM for safety.

Parts safety

During our project, 7 different genes were assembled to be cloned in E. coli. The origin of these genes was checked during the design process and, as explained in the iGEM risk group tools, the molecule produced should prevail in terms of safety over the donor organism. Before building our DARPin and Allergen plasmids, the origin of genes was checked, and summed up on the following table (Table 1):

Table 1. Used genes and their donor organisms.

All allergen genes are theoretically harmless since the corresponding protein can be encountered by humans during their everyday life. DARPin is a lab designed protein for medical purposes to treat allergic reactions (Baumann et al. 2010), so the DARPin can be cloned in our lab. During lab experiments, we used classical fluorescence genes, such as mScarlet, BFP, RFP, and sfGFP, and their harmlessness permitted the use in the lab. All genes were supplied by IDT or institutional researchers from our partner laboratories TBI and TWB.

French legislation

Every French lab workers, including all of us must proceed to respect the French legislation, specially Orders of July 16, 2007 and December 27, 2017 fixing the technical measures of prevention, in particular of containment for places such as laboratories of research, analyses and teaching where people are liked to be in contact with biological pathogenic agents (NOR: MTST0756429A and MTRT1633568A). In addition, the decree of March 7, 2008 on the prevention of biogenic risks (Articles R4421-1 to R4427-5), present in the French labor code have to be respected, and more specifically for GMOs, the order of March 28, 2012 impose on us to make a file for contained use of GMOs according to the article R. 532-6, R. 532-14 and R. 532-26 of the environmental code.

Laboratory security

The non-pathogenicity of bacteria does not allow us to forget basic security rules or not to use security equipment available in the laboratory. The security rules, good practice on lab and security materials are listed below:

• - People in the laboratory have to wear a lab coat, appropriate clothes, covered shoes, gloves.

• - The lab has to stay clean and tidy.

• - Personal belongings, drinks and food have to stay outside of the lab.

• - Hands have to be washed regularly, specially between manipulation and breaks.

• - Wastes have to be sorted in appropriate bins, contaminated wastes have to be in a specific bin, sharp objects and glasses in specific boxes, and chemical wastes in the relevant containers.

• - Contaminated solutions with biological products have to be treated by autoclave or with bleach.

• - All container and biological samples have to be labeled.

• - All autoclave procedures were performed according to the formation we received at our entrance in the lab (see below).

• - Fume cabinet was used to manipulate chemical products.

• - A laminar flow cabinet was used to avoid contamination of microbiological experiments.

• - Manipulations with Ethidium Bromide (EtBr) have been done in a specific locked room with materials attributed to this very use, and strict security rules. A specific formation had to be followed. Specific gloves and glasses have to be worn at all times. There are also specific procedures for waste materials and solutions contaminated with EtBr.

With the help of our lab security manager, we have listed chemical products which need special attention during manipulation (Table 2).

Table 2. Used products and their associated dangers.

Safety formations

Our project was performed under the responsibility of the Toulouse Biotechnology Institute (TBI) and our home engineering school INSA Toulouse. For this reason, they offered us the necessary formations to insure our security and the safety of the premises and of the environment. The first week of lab practice were dedicated to raise awareness in the team to security:

• - We visited the lab on the first day with our principal instructor explaining to us where the dangers are, the positions of extinguishers, security showers, security eyes-washers and emergency exits.

• - We used Neo, a e-learning platform for all newcomers to institutional laboratories in France. This numeric support contains interactive resources such as videos, sentences with gaps, questionnaires, and learning games to efficiently learn important security aspects. At the end, the TBI safety manager controlled our results and organized a troubleshooting reunion to insist on important points and to answer our specific questions.

• - Manipulations with bacteria required sterile materials and decontamination. We were formed to use the autoclaves by the material manager. He explained to us the risks linked to the use of the two autoclaves available in our laboratory, and showed us how this works. Most of the team was habilited to using the autoclave. During the first sterilization operations, we were assisted by our lab technicians for troubleshooting, checking with us critical operations and giving us their recommandations.

• - The PI has specifically taught us how to use the EtBr room to reveal gels and manage EtBr baths and wastes safely for us and for the laboratory.

For troubleshooting and checking if manipulations need specific security rules, lab technicians and our instructors were present to direct us during the whole summer.