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Safety

iGEM Questioniare

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

In terms of biological safety, the project ideas of the TU Munich Team 2012 do not raise any more issues than those which have to be considered in every biotechnological work involving genetics and microbiology. However, since the project aims at applications in food and nutrition, the possible effects of the compounds we intend to produce must be considered. Also, the application of genetically modified organisms (GMOs) in food production in general is relevant for this project and will be discussed here.

A regular safety briefing and a lecture about the legal basics concerning biotechnology and genetic engineering are basic elements of our education at the TU Munich. Here, the handling of biological material, aspects on chemicals and the circumstances and protocols at the lab we work in (these may differ from institute to institute) are explained. Even though most of us have worked in laboratories before, there are some aspects which need to be repeated before 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, what kind of biological parts and chemicals he is using 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. This classification of the respective Biosafety levels is very similar to the one given in the World Health Organization (WHO) Laboratory Biosafety Manual.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. All parts used in our project are either derived from microorganisms or plants (except fluorescent proteins). During our labwork, we used the following microorganisms:

• Escherichia coli, strain XL1 blue (a K12 strain, used for molecular biology applications, i. e. cloning and amplification of plasmids)
• Saccharomyces cerevisiae, strain INVSc1 (used for molecular biology applications, i. e. characterization of parts and as a donor of genomic DNA for isolation of promoters)
• Saccharomyces cerevisiae, strain Y190 (used for molecular biology applications, i. e. characterization of the light-switchable promoter system)
• Kluyveromyces lactis, strain WSYC115 (used as a donor of genomic DNA for isolation of the KlADH4-promoter)
• Spirulina platensis, dietary supplement (used for organic chemistry applications, i. e. extraction of phycocyanobilin)

The German Federal Office of Consumer Protection and Food Safety (“Bundesamt für Verbraucherschutz und Lebensmittelsicherheit, BVL) regularly publishes a list of legal classifications and risk groups of microorganisms (according to §5, Section 6, GenTG). In this list, E. coli K12 strains,S. cerevisiae and K. lactis are classified as BSL 1 (biosafety level 1). Spirulina platensis was not used in form of vital microorganisms but as a powder which is commercially available as a dietary supplement which has not shown toxic effects during toxicological studies (1).

The following table includes all organisms from which parts we used are derived, but which were not used in the lab (donor organisms):

According to § 4, Section 1 (GenTG), the properties of the donor organism itself have to be considered if the donor organism is actually used during experiments. However, we obtained the DNA for these parts either from other research institutes or had them synthesized. This means we never used these organisms ourselves and therefore, only the properties of the genetic material we used must be considered regarding safety. All the parts listed above are genes coding for proteins/ enzymes which are either involved in a biosynthetic pathway or have a desired property and are not hazardous (BSL1).

Just like in every other biochemical laboratory, there are substances and devices in our lab, which are potentially dangerous.

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. The substance we use is ethidium bromide. To prevent skin contact, protective gloves should be made from nitrile rubber and changed frequently to prevent contamination ethidium bromide. All gels and materials that came into contact with ethidium bromide 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 hydrochloric 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.

For the extraction of phycocyanobilin from spirulina powder, we also performed some basic organic chemistry experiments. In particular, we carried out a methanolysis, which involves the application of Methanol and Chloroform in significant quantities (up to 500 ml of Methanol and 50 ml of Chloroform). These two substances can have damaging effects on the health of the experimentator and must be handled carefully. Also, when using equipment such as reflux condensers or rotary evaporators, the experimental setup must be checked carefully to prevent breaking of glass and other accidents.

Our project aims at using genetically modified organisms (GMOs) for production of beer. In Germany, using GMOs for production of food is viewed very critically by the general public. Many people are afraid of possible risks and long term effects on ecosystems if GMOs are released to the environment. Although food additives such as the flavor enhancer glutamate are routinely produced with GMOs in industry, the general public oftentimes does not differentiate between food ‘’containing’’ GMOs and food containing substances ‘’produced’’ by GMOs within a facility and might therefore have objections against it. Because of this, the “Staatsbrauerei Weihenstephan”, which is the world’s oldest brewery and located on our campus, asked us to emphasize the fact that there is no relation between our iGEM project and the “Staatsbrauerei Weihenstephan”. This shows that using GMOs for production of food is a very sensitive topic. As a consequence, a member of our team talked to an employee of the German Federal Office of Consumer Protection and Food Safety (BVL) during the CAS SynBio Conference which took place from July 23rd -25th near Munich. During this conversation, the representative from the BVL said that he did not see any concerns regarding our project, because the substances we wish to produce are safe in foodstuffs and because the genetically modified yeast can be removed from the beer by filtering. Filtering is a normal step in the process of brewing and can guarantee that all GMOs are contained within the facility. Preventing the release of GMOs was a key point in the planning of our project and one of the reasons why we chose brewing as application for our project. Additionally, we organized a get-together of politicians from several political parties and experts on the field of GMOs mid-september (see our human practice section) to discuss our project and GMOs in nutrition in general.

The microorganisms we used are not motile and auxotroph, so they cannot survive in minimal medium (with only glucose as Carbon 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 cultures of microorganisms and waste containing biologic material is autoclaved at 121 °C before being thrown away. This ensures that no genetically modified material is released from the lab. Our final constructs themselves, the various promoter systems and biosynthesis pathways from plants, are not associated with pathogenicity, infectivity or toxicity. Furthermore, they have no impact on environmental quality, as it are unable to compete with its natural competitors, due to its auxotrophies. All in all this leads to secure strains which cannot survive outside the laboratory. Also, a deliberate misuse of our constructs is implausible. The newly introduced genetic material is derived from natural pathways found in plants and does not increase pathogenicity, infectivity or toxicity. This is also true for all construction intermediates.

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

None of the biobricks we produced this year are harmful to humans or the environment. The products that are produced by our transgenic yeast strains during brewing or other applications are caffeine, limonene, xanthohumol and thaumatin. All these substances are found naturally in various foodstuffs. Thaumatin has been approved as a sweetener by the European Union (E957) and as a Generally Recognized as Safe flavoring agent (FEMA GRAS 3732) in the United States. There are hazard indications for the substances caffeine, limonene and xanthohumol according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS), but the concentrations which can be produced with our system are far below any critical concentrations and within the range of concentrations which are naturally found in foodstuffs.

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

Every department of the TU Munich needs a safety delegate, in our case (NAME?). In a direct conversation about our project, he/she did not mention any concerns with regard to 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).

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 microorganisms 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.

References

[1] Salazar, M., Martinez, E., Madrigal, E., Ruiz, L.E. & Chamorro, G. A. (1998), ‘ Subchronic toxicity study in mice fed ‘’Spirulina maxima’’, ‘’Journal of Ethnopharmacology‘‘ Volume 62, Issue 3,Pages 235–241.

Bavarian Purity Law