The safety regulations involved in our experiments are crucial for protecting not only the team members but also the surrounding community. Therefore, in addition to following the standards established by iGEM and the biosafety authorities of our institution, we consulted with experts in this field to guide us and assess the risks and relevant safety issues related to the development of our project.
Safety in the Laboratory
Safety in GMOs production
One of the pillars of our project is the production of antimicrobial peptides in genetically modified microorganisms. In this context, we chose Escherichia coli DH5-alpha BL21, Saccharomyces cerevisiae BY4742, and Aspergillus oryzae ORY7 as the chassis organisms for our project. The manipulation of these microorganisms was carried out in biosafety level 2 laboratories, meeting all requirements for sterility, biological waste disposal, and the use of personal protective equipment as required by our institution, UNICAMP.
Safety in Phytopathogen Testing
To test our antimicrobial peptide CTX, with the aim of addressing the diseases that affect citrus crops, we worked with the phytopathogens responsible for Huanglongbing (HLB/Greening), Green Mold, and Sour Rot.
The pathogen causing Greening is Candidatus Liberibacter asiaticus, a non-culturable bacteria that inhabits the phloem of citrus trees. Due to the impossibility of propagating this bacterium under laboratory conditions, our assays were performed using citrus leaves already infected with Greening. The handling of this material was conducted exclusively in a greenhouse specialized in the study of this phytopathology, under controlled conditions.
The pathogen responsible for Sour Rot is Geotrichum candidum, a filamentous fungus found in soil, decaying organic matter, and on the surface of fruits, particularly oranges. As this pathogen is considered opportunistic in humans, it is classified as a Biosafety Level 2 (BSL-2) organism. The manipulation of this microorganism was carried out at the LaBioQuiMi laboratory (UNICAMP), which specializes in its study, under controlled and sterile conditions.
The pathogen responsible for Green Mold is Penicillium digitatum, a filamentous fungus with a high sporulation capacity, capable of producing billions of conidia per fruit, which facilitates its dissemination under humid climates. Although generally considered non-pathogenic to humans, P. digitatum is classified as a Biosafety Level 2 (BSL-2) organism due to its potential to cause allergic reactions or opportunistic infections in immunocompromised individuals. The handling of this microorganism was performed at the LaBioQuiMi laboratory (UNICAMP), the same facility where we conducted the G. candidum assays, under the same controlled and sterile conditions.
Safety in AMPs Testing
The experiments conducted with the antimicrobial peptide (AMP) from our project, named CTX, were carried out under controlled conditions in Biosafety Level 2 laboratories. Although CTX has not been extensively tested for its potential effects on human health, we considered the possibility that it could exhibit cell-disrupting activity. Therefore, we strictly followed all institutional regulations for working with potentially hazardous molecules. The peptides were applied exclusively in in vitro microbial inhibition assays, and no ingestion or toxicity tests were performed during the project. Consequently, no direct contact with CTX, whether purified or not, was made with any animal or human.
Safety in Human Practices
As part of our Human Practices activities, we developed a public perception survey to better understand the general public’s knowledge of topics related to synthetic biology.
For the publication of the survey results, we obtained consent authorization from all volunteers who responded to our questionnaire. The answers were analyzed in general terms, based on the percentages of the selected alternatives, ensuring the anonymity of participants in the dissemination of the results.
How did we learn about biosafety regulations?
Throughout the development of our project, biosafety was always a priority, both during on-site visits to orange groves and in the execution of laboratory experiments. To ensure this, we conducted in-depth research that provided us with a clear understanding of the necessary regulations for conducting our experiments, including the handling of transgenic organisms and pathogens, disposal of biological waste, and good practices for maintaining sterility in the experimental environment, which were briefly described earlier. In addition, we sought guidance from specialists at our university who could assist us in applying current biosafety regulations, as well as advise us on which institutional bodies to contact in case of laboratory accidents.
As we are part of a Brazilian university, we follow the biosafety regulations established by Law No. 11.105/2005, which defines safety guidelines and oversight mechanisms for the handling, transfer, and use of GMOs and their derivatives. In this context, we spoke with Dr. Welbe Oliveira Bragança, manager of the Internal Biosafety Commission (CIBio) at UNICAMP, the body responsible for ensuring compliance with these regulations within the university. Under his guidance, we received support to maintain a high level of biosafety throughout the project and gained a clear understanding of the containment protocols applied in facilities that work with GMOs.
For the genetic manipulation of Escherichia coli, Saccharomyces cerevisiae, and Aspergillus oryzae, we relied on the training provided by our PI specialists, Dr. André Damásio and Dr. Elizabeth Bilsland, who ensured that our experiments were conducted safely and under controlled conditions. They also provided all the necessary infrastructure for carrying out our work in their laboratories, LEBIMO and LaBS (UNICAMP).
Furthermore, as mentioned previously, for the handling of pathogenic fungi, potentially hazardous to humans, we received instruction from Prof. Dr. Taicia Pacheco Fill, coordinator of the LaBioQuiMi laboratory (UNICAMP), specialized in the study of these microorganisms.
Finally, the experimentation with Greening-infected plants was guided and supervised by Prof. Dr. Alessandra Alves de Souza, a researcher at the Instituto Agronômico de Campinas (IAC), specialized in the study of citrus phytopathologies.
