At its core, our project is to generate and characterize functional variants of three promoter/repressor systems that are commonly used in the parts registry. This involves the use of standard molecular biology techniques (i.e. mutagenic PCR, cloning, and GFP expression). Simple devices will be built from combinations of our parts to demonstrate the utility of our new parts library.

All parts in the BioBrick system are placed into backbones containing antibiotic resistance genes which aid in the culture and selection of bacterial strains during the culturing process. These genes pose some risk to the environment in that there is a possibility that they could be transferred to local bacterial populations. For this to occur, strains containing antibiotic resistance genes would need to escape the controlled environment of our lab and to survive and grow.

We take protective measures to prevent this from happening. All plastic materials that come in contact with bacterial strains are autoclaved thoroughly prior to proper biomedical waste disposal. All liquids that might contain resistant cells are bleached before disposal. Lab workers do not wear gloves when outside the lab to avoid transmission of these bacteria to other parts of the facility.

If somehow our strains were introduced into the outside environment, it is unlikely that they would do serious damage. The antibiotic resistance genes offer very little selection advantage outside of a laboratory setting, and it is unlikely that strains would out-compete other bacterial strains. Our strains contain no islands of pathogenicity or other genes that would cause them to pose a threat to animal or plant populations outside of the lab.

Our project poses no more public safety concerns than any other iGEM project. The strains of E. coli we work with are non-pathogenic and considered safe, standard laboratory organisms. They do not possess any resistance to antibiotics not conferred by BioBrick backbones. Parts included in our constructs do not code for any toxins that would pose a threat to people. Our lab is not accessible to the public, and no materials are removed from lab without proper containment.

All personnel working in our lab are familiarized with our safety protocol regarding hazardous chemicals, biological waste, and mechanical hazards around the lab. They are required to sign safety forms to acknowledge their understanding of safety procedures and expectations. They are familiarized with lab safety equipment such as eyewash stations, emergency showers, spill cleanup kits, and first aid kits.

Our lab is kept clean, with minimal clutter on lab benches. This reduces the risk of chemical spillage and fire. When possible, we take effort to use chemicals that pose low risk to our researchers. For example, instead of using Ethidium Bromide to stain our gels, we use an alternative called SYBR Safe that poses much lower health risks. When dangerous chemicals must be used, we have several fume hoods available to researchers to minimize aerosol exposure, and designated containment areas for flammable, corrosive, and otherwise hazardous reagents. Researchers are required to wear face and eye protection, a labcoat to minimize skin exposure, and rubber gloves whenever working with ultraviolet light.

1.Would any of your project ideas raise safety issues in terms of:

• Researcher safety? No
• Public safety? No
• Environmental safety? No

2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? how did you manage to handle the safety issue? How could other teams learn from your experience?

• Our parts are all variants of commonly used repressor/promoter pairs that are already present in the Parts Registry. None of the existing parts have been previously shown, on their own, to pose any safety hazard. Consequently, it is reasonable to expect that none of our mutant parts will raise any additional safety concern. More general concerns related to any BioBrick part could be to minimize the spread of antibiotic resistance genes, encoded in all BioBrick backbone vectors, into the environment. While unexpected, if we do discover that any part used in our project is potentially dangerous, we will note it on the Registry page and take extra precaution when handling strains of bacteria containing constructs which include said part.

3. Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project?

• Our institution has a biosafety committee that inquired about and reviewed the scope of the work conducted by UC Davis iGEM students. There were no concerns reported after their committee discussion. We also have a local Biosafety Officer whose office is 30ft. from our laboratory. She is, obviously, aware of our project and provided we follow local safety protocols and guidelines (which we do) is also supportive of our work. The facility in which we work is a designated center for biomedical and genetic research and therefore provides some key structural safety measures required for working organisms up to BSL2. None of our work, or the work in our host lab, exceeds BSL1 activity. We also follow pre-existing health and safety protocols, including the proper use of personal protective equipment and use of designated biotechnological waste disposal stream as required by UC Davis and our host lab.

4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?

• We think that instituting a standardized set of protocols for testing and characterizing the behavior of each part in different contexts may be important for understanding if a part and/or device poses an unexpected safety risk. For instance, last year's UC Davis iGEM team discovered that a commonly use part C0051 (the lambda cI repressor) actually had relatively strong promoter activity. This was, as it turns out, due to a bar code that was added by the registry with the intention of providing a means for tracking the origin of parts if they were found used in an inappropriate manner. In our case, this function was discovered by accident, during the construction of a polycistronic transcript in which the gene encoding RFP followed C0051. Had RFP actually been a gene producing a potential toxin rather than RFP - even if the overall design of the device was safe - the uncontrolled expression of said toxin may have posed safety risk. Having a standard screen for all new parts for promoter activity, for instance, might provide more data on the behavior of the parts. This in fact was one of the motivators for our project this year - providing some detailed characterization of parts: this will not only help us understand how the parts will work under various conditions, but should also reveal potential safety concerns.
  
  Responsibility for providing details about the safety concerns for a given part should fall first on the team responsible for submitting it to the registry. A greater emphasis on teams submitting well-characterized parts would not only allow said parts to be used more efficiently, it would also reduce the risk of unsafe parts being widely distributed without the risks of their use being well-known.