Answers to the iGEM Safety Questionnaire:

1. Would any of your project ideas raise safety issues in terms of:
- researcher safety,
- public safety, or
- environmental safety?

A: No, as long as safety guidelines and practices as detailed below in the text are followed. All work was performed under the approval of a Biological Use Agreement (BUA) approved by UCSF Environmental Health and Safety (EH&S) and all personnel were trained and certified for the work.

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?

A: No, as detailed below in the text, all of the devices / parts are considered BSL1. Only the human cell lines are considered BSL2. Safety concerns and procedures that we considered during running the project are found in the text below.

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?
- If no, which specific biosafety rules or guidelines do you have to consider in your country?

A: All work was performed under the approval of a Biological Use Agreement (BUA) approved by UCSF Environmental Health and Safety (EH&S), and all personnel were trained and certified for the work. Our iGEM team discussed the project with the host laboratory’s safety officer and were informed that the work was covered and approved by UCSF EH&S under the host laboratory’s BUA.

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?

A: One potential idea is described in the text below. This would be to incorporate a suicide gene into the final constructs. This may be more important if the devices raised safety concerns or if they were incorporated into viral delivery systems.

Description of Our Team's Safety Practices

The safety of the UCSF 2010 iGEM team, other scientists in the shared lab area, and environment has been a great concern for us throughout the project. This required extra precautions and training as our project used human cell lines which are handled under Biological Safety Level II (BSL2) guidelines. Before we were even allowed to enter the labspace, every member of the team was certified by UCSF’s Office of Environmental Health and Safety (EH&S) after completing the required lab safety training. We also received extra training on tissue culture guidelines, rules, and safety by Krista McNally who manages the Cell Propulsion Lab tissue culture facility. Throughout our project, we wore personal protective equipment (PPE) such as lab coats and gloves while working in the lab area and strongly adhered to the safety guidelines of the Office of EH&S. The DNA cloning and bacterial work was performed on the lab benches under Biological Safety Level I (BSL1) guidelines. A separate lab coat and new gloves were used when working in the tissue culture room to prevent cross contamination between the bio-hazard levels. Ethanol was used in the tissue culture room and on tables to kill any microbes that might be on the surfaces to prevent contamination. Dilute bleach (10%) was also used to treat liquid cultures prior to disposal and solid materials were placed into bio-hazard bags in marked containers for disposal. We learned that being organized and cleaning up before and after procedures was a good safety practice that also assisted in keeping our cultures from being contaminated.

During our iGEM project, we attempted to substitute procedures and chemicals/reagents that would minimize risk. As an example, for running our DNA gels we used SYBR Safe dye rather than using ethidium bromide which has risks from mutagenic potential and whose use generates hazardous waste that leads to disposal concerns. As another example, we used electroporation to deliver plasmid DNA containing our devices into our human killer cell lines. This was ultimately a disadvantage to us as the efficiency of gene delivery/expression was low, but we did not consider and were not approved for using higher risk methods of gene delivery into these cells such as viral delivery, which has been shown to be more efficient but at higher risk. All of our new devices and parts that we made for our 2010 iGEM project are plasmids, which did not raise the level of safety concerns that infectious agents such as recombinant viruses would.

We believe that as long as the proper precautions are taken and the safety guidelines are followed, most potential safety concerns can be prevented. None of our genes, parts, or devices are considered potentially oncogenic or pathogenic which would require a safety rating above BSL1. We specifically chose not to use materials from known pathogens. A possible extra precaution to make parts, devices, and systems safer would have been to put suicide genes into the sequences to prevent unintended introductions of them into the environment, but this was less necessary based upon the components and systems we used.