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 need to be reminded of when starting to work in a lab. The researcher should wear a labcoat, safety glasses and gloves and one must not drink, eat or smoke whilst working at the bench. The safety degree of the worn protection should depend on the chemicals and microorganisms handled. The most important part, however, is that everybody should always be aware of what he is doing, with what kind of biological 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 potentially harmful to the researchers if they act corresponding to the general precautionary measures. Especially, no pathogenic organisms are used, as the bacterial strains in our lab do not possess mechanisms necessary for survival outside of the lab, or in the human body. However, even in a BSL 1 lab materials are used, which can be harmful to researchers, the public or the environment.

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 which 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 out of the lab, or harm its researchers.

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?

There are dangerous substances used in our lab. The following list features a few important examples:

1. In every laboratory of molecular biology, specific chemicals are required for staining of DNA, in order to make it visible on Agarose gels. Most of them directly intercalate into the double strand of DNA, which makes them cancerogenic. A commonly used, but rather dangerous substance is ethidium bromide. In our lab, we use SYBR® Gold. It is less hazardous than ethidium bromide, but can still be cancerogenic if it comes into direct contact with human skin. Protective gloves should be made from nitrile rubber and changed frequently to prevent contamination with SYBR® Gold. All gels and materials that came into contact with SYBR® Gold need to be disposed of seperately. This is done in order to prevent their unintended leakage into the environment, with subsequent harm to humans, animals and plants.
2. Methods of molecular biology often require strong acids or bases, like sulfuric acid, or toxic substances such as methanol. They need to be handled with extreme caution and also need to be seperately disposed.
3. Many devices in the lab can be potentially dangerous towards researchers, if they are used carelessly or in the wrong way. There are lamps emitting ultraviolet radiation, which can be cancerogenic.

If all these measures are taken, the potential danger for researchers, other people and the environment can be reduced to a minimum.

There are several different bacterial strains used in our lab for transformation of BioBrick parts, which have been manipulated in some way to make sure that they are harmless. The strains used in our lab, like the heat-resistent Escherichia coli strain BH28, are all derived from E. coli K-12. K-12 is a safety strain, as the bacteria carry several auxotrophies. This means that they are dependent on certain sources of carbon, amino acids and other nutritients. Without them, growth is not or only in a restricted way possible. BH28 is derived from K-12, but has over 260 mutations, which lead to its heat resistance. The E. coli strains DH5 alpha, BL21 and CP919 are also derived from K-12.

If despite all precautions humans are infected with bacteria, the first weapon of choice are always antibiotics. They specifically kill bacteria, while keeping side effects for the patient to a minimum. Bacteria can be resistant against antibiotics. The genes responsible for this are often used as selection markers during cloning. With respect to killing them however, antibiotic resistences can make bacteria more dangerous. The number of supplied resistences should therefore be reduced to no more than necessary.

The bacteria are not motil and auxotroph, so they cannot survive in minimal medium (with only glucose as C source), but need additional aminoacids to survive. Since nothing from the lab is taken into public and stays inside there should be no safety issues considering public or environmental safety. Furthermore, used E. coli cultures and waste containing biologic material is autoclaved at 121 °C before being thrown away. This ensures that no genetically modified material can reach the outside of the lab. Our final construct itself, the optogenetical AND-Gate, is not associated with pathogenicity, infectivity or toxicity. Furthermore, it has no impact on environmental quality, as it is not able to compete to its natural competitors, due to its auxotrophies. All in all this leads to secure strains which cannot survive outside the laboratory.

A deliberate misuse of this construct is unplausible, as it does not increase pathogenicity of the E. coli strain. Scenarios, in which our construct is used to specifically trigger the production of toxins or other substances to harm humans, animals, or the environment in general are unlikely, as the strains used are not able to survive outside from controlled conditions. Even the use of efficient strains would not be able to promote misuse, e.g. as a biological weapon, because the optogenetical AND-Gate does not feature necessary factors of pathogenicity. 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 potentially harmful to humans or the environment. The function of the optogenetic AND-Gate is to control the expression of a protein by light. The used reporter plasmids express β-galactosidase or GFP and both do not pose a threat to humans, animals or the environment.

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 this is Guenther Woehlke. In a direct talk, he did not mention any concerns about the project regarding safety issues. Also, Helene Budjarek, the supervisor of safety in the lab, sees no possible risks 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).

Before starting work in a lab, every participient has to get a safety instruction and an instruction in the lab, which were both obligatory. Our supervisor of safety, Helene Budjarek, made us familiar with the lab and explained the safety rules when handling with dangerous biological or chemical substances (e.g. SYBR® Gold) or devices. She also showed us how to properly act in an emergency and where to find help, like showers or fire extinguishers. Fore more information, also refer to section 1 above.

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?

There are several ways to increase safety when working with BioBrick parts. The easiest way is to protocol and supervise all actions, parts and bacterial strains during the creation of new, or the improvement of existing BioBricks. By denying unauthorised people access, misuse can be prevented. Furthermore, the bacteria need to be kept dependent on controlled conditions in the lab, in order to prohibit their uncontrolled spreading outside the lab. The uncontrolled multiplication and spreading of parts must also be avoided. This can be achieved by application of uncommon restriction enzymes and by using no parts containing infectious DNA in combination with parts that can multiply and spread without the help of a host organism, like transposons.