PPROMNESIS project operated with organisms permitted at the EU Biosafety Level 1 (BSL-1) —Escherichia coli, and EU Biosafety Level 2 (BSL 2) - BV2 mouse microglia, HEK 293, DH5α, Bacillus velezensis (CVM 168). All experimental work took place in certified bio-safety level 2 facilities. Before entering the lab, every team member completed a compulsory safety training. All procedures were performed in line with applicable European regulations and of the Insitute of Biochemostry of the Romanina Academy internal protocols.
Laboratory Organisms and Facilities
Genetic Parts and Antimicrobial Control
Every genetic part we used or designed was either on the official iGEM whitelist or received authorization through the appropriate application process. Aware of the risks inherent to laboratory research—particularly the emergence and dissemination of antibiotic resistance—we applied strict antimicrobial practices and used antibiotics only as permitted under safe-use guidelines. We also took appropriate measures to mitigate the risks associated with using chemical stress inducers, such as tBHQ, tunicamycin, thapsigargin and IL-6 cytokine.
A summary of PROMNESIS’ safety features
The Logic Gates module allows for control of therapeutic release in the brain. By only allowing for the production of the theraputic when 3 distict AD biomarkers are present, the logic gate module allows for increased saftey and reduction of adverse effects of the theraputic system, by only allowing the therapeutic to act on cells affected by AD pathology.
In TauTuner, an optogenetic restriction – insured by the presence of LOV2 domain, limits the activity of the therapeutic, thus offering the possibility of external control. In practice, this is tested under lab-controlled light boxes.
TRI-LYTACensures physical containment during delivery of therapeutic cargo. The PEGylated liposomes constitute a physical barrier and are being degraded in a short period of time
Responsible Use of Artificial Intelligence Tools
We identified the risks associated with using AI tools, such as AlphaFold for structural modeling (e.g., LOV2–PP2A fusion proteins) or ChatGPT (e.g. for brainstorming during the conceptualisation phase of the project, by generating lists of literature references). To address the potential inaccurate structural predictions, inaccurate data or irrelevant literature suggestions, we cross-checked, reviewed and selected the AI-assisted outputs. These were utilised as a guide, not as a final decision, which was based on our team’s review and selection of material, and experimental or literature-based verification.
Risk Mitigation and Compliance
Although research can never be entirely risk-free, we implemented robust measures to limit any potential impacts of our constructs on human health and the environment. Our work adhered in full to iGEM safety standards, regulations, and protocols.