Safety

1. Would any of your project ideas raise safety issues concerning researchers, the public or the environment?

The project ideas of the TU Munich Team 2011 do not raise any more safety issues than those which have to be considered in every biotechnological work involving genetics and microbiology.

All team members had to participate in a safety briefing, where we learned handling biological material, aspects on chemicals and the circumstances and protocols at the lab we work in (these may differ from institute to institute). Even though most of us have worked in laboratories before, there are some aspects you can’t hear enough of. In general, the researcher should wear a labcoat, safety glasses and gloves and one mustn't drink, eat or smoke whilst working at the bench. The most important part, however, is that everybody should know in every step of his work, what he is doing, with what parts and chemicals he is working and how to handle them safely.

The lab we work in is classified as BSL 1 (biosafety level 1), according to the European Union Directive 2000/54/EG and the German "Gesetz zur Regelung der Gentechnik (GenTG)" ( law for the regulation of genetic engineering , text in German only). There is a total of four Biosafety levels, with BSL 1 being the lowest and BSL 4 being the highest.

Work inside a BSL 1 lab, such as ours, involves no devices that are harmful to the researchers if they act corresponding to the general precautionary measures. Especially, no pathogenic organisms are used.

BSL 2 and 3 laboratories are necessary to work with biohazardous material that can cause disease (BSL 3 in case of potential severe disease) for wich an effective cure is available. The security measures of these labs include security work benches and an air filter system, for example.

BSL 4 laboratories are required to work with organisms capable of causing severe disease and for which effective treatment is not possible (such as smallpox or the Ebola virus). A BSL 4 lab has to have a broad range of safety measures. To give some examples there have to be hazmat suits, airlocks to maintain a low air pressure inside the lab, and many methods of decontamination to ensure that no traces of biohazardous material can get outside the lab.

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

1. Risks to the safety and health of team members or others in the lab?
2. Risks to the safety and health of the general public if released by design or accident?
3. Risks to environmental quality if released by design or accident?
4. Risks to security through malicious misuse by individuals, groups or states?

The most harmful substance in the our lab is CyberGreen which is used for staining agarosegels after DNA digestion and separation (used a lot in cloning steps). Here everybody has to be careful, switch gloves everytime he touched something containing CyberGreen and in general be responsible and tidy when working with CyberGreen.

Strains:

The E. coli strain MG1655 is derived from E. coli K-12 which is modified so it is not harmful to

Furthermore the strain differs from K12 in 260 more mutations which leads to its heat resistance.

humans. The strain is resistant to kanamycin (by inserted mutation) and ampicillin (during evolution).

The E. coli strains DH5α, BL21 and CP919 are also derived from K-12.

The bacterias are not motil and auxotroph, so they cannot survive in minimal medium (with only glucose as C source), but need additional aminoacids to survive. All in all this leads to secure strains which cannot survive outside the laboratory. Since nothing from the lab is taken into public and stays inside there should be no safety issues considering public or environmental safety.

Used e.coli cultures and waste containing biologic material is autoclaved before throwing away. This ensures that no genetically modified material can reach the outside of the lab. Our finished construct itself, the optogenetical AND-Gate, is not associated with pathogenicity, infectivity or toxicity. Furthermore, it has no impact on environmental quality or raises any security concers. Its only purpose is to control gene expression in immobilized cells in a spatiotemporal manner.

A deliberate misuse of this construct is not possible. This also applies for all intermediate constructs.

3. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?

None of our BioBrick parts are harmful to humans or the environment. We are working with the red-light sensor and the blue-light sensor combined in an AND Gate. None of those parts should survive outside the lab.

4. Is there a local biosafety group, committee, or review board at your institution?

Every department at TU Munich needs a safety delegate, in our case Guenther Woehlke: He doesn't have any objections against the project due to safety issues. Also, Helene Budjarek, the supervisor of safety in the lab, sees no risk caused by our project

We do not have an Institutional Biosafety Committee; all checks concerning safety in laboratories are taken care of by state officials.

In general, working with genetically modified organisms in Germany is regulated by the "Gesetz zur Regelung der Gentechnik (GenTG)" (law for the regulation of gentetic engineering, see above).

To be allowed to work in our lab training is necessary. In our case our supervisor of safety, Helene Budjarek, showed us around the lab and explained the rules of these labs. She also showed us how to work with certain materials (e.g. cyber green) and where to find help, like showers or fire extinguishers, in case of emergency (also see answers to question 1).

5. 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?

The easiest way to increase safety when working with BioBricks is to prevent the uncontrolled multiplication and spreading of parts. This can be acchieved by...

1. ... the use of uncommon restriction enzymes
2. ... not using parts containing infectious DNA in combination with parts that can multiply and spread without the help of a host organism (transposons,…)