1. Would any of your project ideas raise safety issues in terms of researcher safety, public safety or environmental safety?
For our project, the only live organism we are using is the K12 MG1655 strain of E. coli, and all of our parts are also derived from this organism. This organism falls under Biosafety Level 1. Neither our strain of E. Coli nor the genes (aldB, frmr, xylr) are dangerous to humans, publics, or the environment. Only a very few strains of E. coli are dangerous, such as the O157: H7, but our particular strain, K12 MG1655, is not dangerous. Our strain is only capable of causing a mild stomach ache if ingested. E. Coli is also very common in the environment since it is found in the fecal matter of animals. However, soil, sand, and sediments in the environment can naturalize the E. coli. In our lab, we have an autoclave, incubators, Bunsen burners, and a thermocycler which can reach very high temperatures and is handled with extreme care. Only members of the faculty are permitted to use an autoclave, and although our lab is fitted to be a Level II lab, the students only use Level I materials and handle Level I organisms. Ethanol and bleach may damage the skin, so latex gloves should be used when handling these substances. In addition, ethanol is very combustible. We address these problems by always wearing proper safety equipment (lab coat, goggles, gloves, etc.) while being very careful around all laboratory equipment. Horseplay is not tolerated. Mark Adame, our faculty sponsor, requires all team members to read the CDC requirements and regulations for a Level I lab while showing them how to use Level I lab equipment. No harm to humans, publics, or the environment would occur if containment of the E. coli was lost. The risks from malicious misuse are very small; the only harm (a mild stomach ache) from E. coli would occur only if it is ingested.

2. Do any of the new BioBrick parts (or devices) that you made this year raise safety issues?
Unfortunately, due to the lack of funding, our team was unable to submit any standard biological parts, we but are close to finalizing biobricks that we will submit to the registry in the near future. We currently have three theoretical constructs of devices that pertain to three genes in E. coli that detect respective volatile organic compounds.

3. Is there a local biosafety group, committee, or review board at your institution?
There is no official Biosafety Committee or equivalent at our school. However, the team has followed all of the national regulations written by the CDC for a Level 1 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?
In the future, we plan to create a biosafety review board at St. Mark’s to deal with any potential safety issues. This board will consist of the most knowledgeable and responsible faculty members who will know how to deal with any biosafety hazards and make decisions regarding the safety of procedures for the environment, the community, and the researchers.

In humans, E. Coli is isolated in the intestines where it helps with the digestive process; however, when ingested or inhaled, E. Coli can cause severe sickness and, in some cases, hemolytic uremic syndrome, a severe form of kidney failure. To prevent this outcome, many lab safety techniques and guidelines are used.

Before dealing with the E. coli, students will wash their hands thoroughly with water and soap. Afterwards, the students will put on their gloves, goggles, and lab coats. Before we can experiment with E. Coli, we must grow it. The first step of growing bacteria is sterilizing all equipment that will be used which includes pipettes and petri dishes. We also place the petri dishes in autoclaves to ensure that all other microorganisms are eliminated.

Afterwards, we sterilize a test tube with alcohol and then fill it with heated agar, which we pour into the petri dish. For inoculation we sterilize the inoculating loop with bleach and then heat it. We then use the inoculating loop to extract E. Coli from another dish and place it into the agar petri dish using the streaking method. We streak the agar petri dish multiple times with the inoculating loop, heating, sterilizing, and picking up more E. Coli each time. Then we will take the bacteria and incubate it in an incubator. After dealing with the E. coli the students will take their gloves and throw them away into a special bin for gloves and wash their hands thoroughly again.

In the lab we have a fire extinguisher, fire alarm activators, and fire blankets dispersed widely for easy access in case of a fire. In addition, if students accidentally get dangerous chemicals in their eyes or on their bodies, they can run to a shower where multiple gallons of water will be dumped on the student and sprayed into the student's eyes.