Safety
Our CALIPSO project aims to improve chemotherapies, to be more specific and efficient while limiting side effects. We will explain in this part how our project has been thought to be respectful of the legislation and safe for the patient.
Choice of our chassis
Our treatment is safe because we made the choice to use liposomes as a chassis, which are not GMOs. They are biocompatible, biodegradable, and have low toxicity. They are currently being injected without DNA into patients and do not pose a risk. The encapsulated DNA template can solely be expressed in a cell-free system that contains regulatory elements orthogonal to human cells.
Classification of the microorganisms
Microorganisms are classified according to the health risks they can present to humans and the environment:
- Containment class 1 corresponds to operations using GMOs from Group I, and for which the risk to human health and the environment is limited or negligible.
- Containment class 2 corresponds to operations using GMOs from Group II and of low risk to human health and the environment.
- Containment class 3 corresponds to operations using GMOs from Group III and of moderate risk to human health and the environment.
- Containment class 4 corresponds to operations using GMOs from Group IV and which pose a high risk to human health and the environment.
During our experiments, we only used microorganisms from Group 1: E. coli Stellar and E. coli BL21 (DE3) strains.
Parts safety
We made sure to check the origin of our parts.
| Gene / Protein | Origin |
| dhdR and dhdO | Achromobacters denitrificans |
| sfgfp | E. coli |
| anti-HER2 nb | Camelid |
| tymp | Homo sapiens |
| Ppnp | E. coli |
| Pertuzumab | Homo sapiens |
| Trastuzumab | Homo sapiens |
| FKB | Homo sapiens |
| FRB | Homo sapiens |
| T7 polymerase | Phage T7 |
All genes were safe (no toxins) and supplied by IDT or institutional researchers from our partner laboratories TBI and TWB.
Cancer lines safety
We worked with the Caco2 cell line originating from the human epithelial. It has been widely used as a model of the intestinal epithelial barrier. The Caco2 cell line is originally derived from a colon carcinoma. One of its most advantageous properties is its ability to spontaneously differentiate into a monolayer of cells with many properties typical of absorptive enterocytes with brush border layer as found in the small intestine. This cell-line can be used in a Biosafety level-1 laboratory as recommended by ATCC (American Type Culture Collection) [1], since it does not represent a direct danger to the manipulator.
Molecules used
5-FU, Tegafur and rapamycin were used respecting security measures indicated by the supplier. As CMRs, these products were stocked in specific containers, and manipulated with precautions by wearing personal protective equipment.
Safety and security of the project are of primary importance for us. First, the use and creation of GMO could always be a risk for the environment, albeit unlikely. A second form of safety equally important to us concerns the experimenters in their workplace. iGEM Toulouse INSA-UPS rigorously follows the French legislation and the recommendations of the National Consultative Bioethics Committee for Health and Life Sciences (CCNE) to accomplish its task in the best conditions.
French legislations
Every French lab workers, including all of us must proceed to respect the French legislation, specially Orders of July 16, 2007 [2] and December 27, 2017 [3] 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) [4], 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 [5].
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, especially 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 containers 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.
- Cell culture and manipulation of cancerous cells were performed in a specific zone of the laboratory to limit contact with microorganisms. Bins containing biological waste were separated and labeled.
With the help of our lab security manager, we have listed chemical products which need special attention during manipulation.
To reinforce safety in the laboratory and help future iGEM teams to safely manipulate cancerous cells, we decided, in collaboration with iGEM team Patras Medicine, to create a safety guide retracing the importance of precautions when performing cell culture, the risks involved and prevention implemented in laboratories
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 was dedicated to raise awareness in the team about security:
- We visited the lab on the first day with one of our advisors 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. All the team was authorized to use the autoclave.
- We were specifically taught how to use the EtBr room to reveal gels and manage EtBr baths and wastes safely for us and for the laboratory. We were also taught to manipulate other CMRs.
