Safety

Researcher safety?
• Because some standard lab materials were hazardous, we carefully followed all safety precautions regarding their handling and use. Specifically, we used dedicated containers for agarose gels containing ethidium bromide, worked in a fume hood when handling mercapethanol, wore protective gear when using UV light for gel excision, disposed of sharps in sharps waste, and wore gloves at all times in the lab.
• All team members satisfactorily completed online biosafety training for performing BSL2 work (http://ehs.virginia.edu/biosafety/bio.training.html) before working in lab, and most team members took our spring semester course on synthetic biology, which included biosafety orientation. At least two people were in lab to ensure safety whenever a procedure was being performed, and most work was done in the presence of the grad students and post-docs with whom we shared the lab.
• We performed our project in a BSL2-certified lab. We used standard lab strains of E. coli, B. bronchiseptica, T7 phage, and BPP phase-locked phage in accordance with our host lab’s safety practices. The majority of our work was at BSL1, and experiments using B. pertussis were conducted within a collaborating BSL2-certified lab that specifically works with the strain.
• All biological waste was disposed of in containers for contaminated waste and subsequently removed by a licensed waste contractor.

Public safety?
• We do not anticipate any threat to public safety. None of our modifications impact virulence, and our reporter gene is non-toxic.

Environmental safety?
• We do not anticipate any threat to environmental safety from our project. None of our organisms or constructs have any known environmental impacts, all were physically contained using safe lab techniques, and our lab strains are unlikely to be competitive outside the lab.

• We do not foresee any safety issues raised by our BioBrick parts or devices this year. None of our constructs code for human toxins or have any predicted environmental interactions, although chronic internal overexposure of our reporter may be harmful. The diagnostic procedure we envision would not involve exposing humans to engineered organisms or gene products.
• We plan to submit phage lysins as BioBricks. Lysins specifically lyse bacterial cells, and would not affect eukaryotes.

• Our project was approved and overseen by multiple faculty members and performed in an active faculty lab which is directly responsible to our local EHS. All team members satisfactorily completed our local biosafety group’s online training course.

• When constructs are predicted to have consequences if released, engineers should use multiple containment strategies in combination, such as physical containment, nutritional containment, orthogonality, and kill-switches.
• Before synthesis or assembly, protein-coding genes should be screened through protein-protein interaction databases for potential non-intuitive interactions, especially if planned for environmental release.
• Tools for algorithmic or predictive assessment of the safety of synthetic constructs should be developed and deployed, rather than solely relying on self-reported/intuitive projections of synthetic construct safety.