Would the materials used in your project and/or your final product pose:

a. Risks to the safety and health of team members or others in the lab?

b. Risks to the safety and health of the general public if released by design or accident?

c. Risks to environmental quality if released by design or accident?

Our laboratory activity is consistent with the NIH “Guidelines for Research Involving Recombinant DNA Molecules” as overseen by our Institutional Biosafety Committee (see question 3). While we handle all laboratory materials that pose a potential safety risk according to standard lab safety protocol and Materials Safety Data Sheets, we took the following steps to ensure that the health and safety of laboratory personnel remained a priority when using certain materials and equipment as outlined below:

Ethidium bromide (EtBr): All EtBr use is strictly performed on a designated bench with separately kept equipment (pipettes, tips, waste receptacle, gloves etc). Any materials that come in contact with EtBr, including gels used in electrophoresis, are handled with nitrile gloves and lab apron and disposed in specially marked receptacle with a “biohazard” designation.

Cell culture: While our bacterial chassis, E. coli DH5α, is disabled to where it is nonpathogenic and cannot survive outside of lab conditions, we exercised standard lab safety protocol to avoid any direct contact with it and associated materials such as antibiotics and exposed tips, plates/broth, and so on. These are all disposed in “biohazard” designated receptacles. Furthermore, all exposed counters are disinfected using 70% ethanol immediately following cell culture and also after each other use of lab. In addition, lab equipment is thoroughly cleaned and autoclaved after use.

UV Transluminator: Use of the transluminator (i.e. during gel extraction) was performed while wearing protective gear such as UV-protective goggles. Further exposure to UV rays and hazardous materials was limited by wearing a long-sleeved lab apron and nitrile gloves.

d. Risks to security through malicious misuse by individuals, groups or states? Access to our building and laboratory is strictly limited by cardkey and is further overseen by security personnel at the building entrance.

2. If your response to any of the questions above is yes:

a. Explain how you addressed these issues in project design and while conducting laboratory work.

See question 1

b. Describe and document safety, security, health and/or environmental issues as you submit your parts to the Registry.

We are working with genes encoding known properties and utilized safety measures to ensure that biohazardous materials including antibiotic-resistant cells are contained within the lab and are appropriately disposed. Therefore, we assess that none of our planned parts raise safety issues and all other documentation will be available on our parts page.

3. Under what biosafety provisions will / do you operate?

a. Does your institution have its own biosafety rules and if so what are they? Provide a link to them online if possible.

b. Does your institution have an Institutional Biosafety Committee or equivalent group? If yes, have you discussed your project with them?

UT Dallas has an Institutional Biosafety Committee (http://provost.utdallas.edu/policy/ utdpp1016) that manages all safety responsibilities under NIH “Guidelines for Research Involving Recombinant DNA Molecules”. Throughout the course of this work, we ensured that all lab activity respected safety measures.

Describe any concerns or changes that were made based on this review.

c. Will / did you receive any biosafety and/or lab training before beginning your project? If so, describe this training.

Our instructors demonstrated use of all equipment and materials in accordance with safety guidelines. This training included an emphasis on wearing protective gear at all times, proper use of large machines such as the autoclave, waste disposal, and specific comments on the use of EtBr-exposed materials.

d. Does your country have national biosafety regulations or guidelines? If so, provide a link to them online if possible.

The NIH outlines specific “Guildlines for Research Involving Recombinant DNA Molecules” (http://oba.od.nih.gov/rdna/nih_guidelines_oba.html), which were strictly observed throughout the course of our project.

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

As part of our project, we are engineering a systemic kill-switch whose induction can be used to regulate the activity of cells harboring our BioBricks. This device will exist in a secondary bacterial population that will confer an additional level of user control. We would encourage fast-acting kill-switches as standard practice in synthetic biology to enable immediate inactivation should significant risks to safety become evident.