Safety

Our project is planned from the ground up to prioritize safety. The only hazardous reagent we handle is ethidium bromide as a fluorescent tag, both our host microorganism S. cerevisea and our procedural intermediate E. coli are benign, the genetic circuit assembled in our recombinant DNA is manifestly non-virulent, our project phase structure minimizes risk to researchers, and many other facets of project planning and execution ensure the highest possible safety standards. We work with exclusively Biosafety Level 1 organisms in a Biosafety Level 2 lab in compliance with departmental biosafety guidelines as well as the NIH biosafety guidelinesfor projects involving recombinant DNA. We are also in close consultation with experienced research advisers and lab managers, and they in turn with UVA’s Institutional Biosafety Committee. Team members underwent semester long lab training in the spring that emphasized safety, our lab manager works full time in our laboratory space, and we’ve carefully reviewed and consolidated our safety protocols.

Hazardous Materials

Specifically to address the carcinogenic risk of ethidium bromide, we strictly follow all lab protocols regarding the use of protective equipment. We store our ethidium bromide stock solution and all dedicated containers in a laminar flow hood, use gloves when handling any of them, and only extract and exponentially dilute microliter-aliquots as needed for gel electrophoresis. Anything that comes into contact with the ethidium bromide is treated as hazardous waste and is disposed of as chemical waste by the in-house DHS.

Risk Analysis

Our device, when working properly, would not represent a virulent hazard to people or the environment. It contains no lethal genes and by nature would be dependent upon a fairly concentrated carbon source. However, over-all system risk is highly dependent on the specifics of output-tuning in that although our engineered organism is non-infectious, one of the growth-factors that it releases to accelerate angiogenesis in wound-healing (VEGF) is also known to contribute to certain diseases (including the growth of tumors) if produced in excessively large quantities. We attempt to mitigate risk in this regard by implementing a feedback control that can be precisely tuned to acceptable levels of output. Risk of unintentional oncogenesis is further mitigated by our selection of the host organism itself because our chosen strain of S. cerevisiea has a doubling time of 2 hours, in stark contrast with standard E. coli doubling times of 20 minutes. This choice yields a much more manageable rate of proliferation in the case of some unforeseen mishap or over-expression.

Environment

Our project does not pose any significant risk to environmental quality if released by design or accident. The circuit generates no known environmentally harmful products. Further, the chassis is dependent on having an abundant supply of a specifically formulated carbon source for nourishment that would be difficult to replicate in the environment. Because the operation of the Gal-1 promoter we’ve chosen is susceptible to inhibition by glucose, we have in lab provided the yeast with a supply of raffinose and galactose to activate the promoter.

Security

Our project does not pose any significant risk to security through malicious misuse by individuals, groups, or states because it is not virulent or a toxin.

Miscellaneous Precautions

Another miscellaneous, low-probability concerns addressed by our project design include the off-chance that our genetic material is lost or intercepted in transit. By opting to use the PCA method for assembly, the genetic material we need to have synthesized offsite is fragmented into single-stranded 60mers, none of which contains functional genetic material, only operational when sequentially combined in the presence of very specific ligases at certain temperatures, which eliminates the risk of spontaneous uncontrolled uptake in the event of an accident during transit in which our synthesized sequences were lost.