"

From 2012.igem.org

Working with genetically modified organismes (GMOs) and microbiology requires safe conditions. The European Union wrote a directive to ensure the safety of the researcher, the laboratory, and the environment. The directive however is very comprehensive and very detailed. The University of Groningen (RUG), also wrote a document listing the rules, regulations, and organizational structures governing work with GMOs at the university. This document is called the ’Administratieve organisatie GGOs’ which is based on the EU directive ‘98/81/EG’ and was approved by the Ministry of Infrastructure & Environment from the Dutch government.

The government is in turn advised by the Netherlands Commission on Genetic Modification (COGEM).
The COGEM provides scientific advice to the government on risks to human health and the environment related to the production and use of GMOs. The organization consists of members appointed by the Minister of Infrastructure and Environment. Each member is selected for their scientific expertise different fields such as ecology, bacteriology, virology, genetically modified plants and animals, zoology, public perception and ethics. More information on COGEM is found on the COGEM website.

Fig.1 Schematic overview from the EU directive ‘98/81/EG’to administratieve organisatie GGOs at the RUG

Students studying microbiology at the University of Groningen are informed on safety aspects of microbiology from the beginning of their study career. They are required to get a VMT (Veilige microbriele technieken, translated: safe microbiological techniques) certificate that demonstrates that they are able to work safely with microbiology and GMOs. The certificate uses, as a foundation, 10 commandments for safe microbiological techniques which are derived from the Administratieve Organisatie GGOs (translated: administrative organisation for genetically modified organisms). Every iGEM member of the iGEM Groningen 2012 team is required to know these rules and uphold them when working in the lab, ensuring a safe work environment for researchers on a daily basis. Most of the team members working in the lab have a background in microbiology, taught at the RUG. These persons have the VMT certificate and know how to work safely in the lab. Team members without a background in microbiology, and who work in the lab, have been instructed with these rules and learned how to work safely.

10 Commandments for Safe Microbiological Techniques

1. All VMT related work can only be performed by those people that have permission from the Biological Safety Officer (BVF). Work according to the rules, even if you believe there is no apparent risk.
2. During VMT related work all doors and windows have to be closed. Verify that insects and other pests are not present in the lab.
3. Wear a closed laboratory coat. Do not take this labcoat outside the VMT area unless it is absolutely necessary for the experiment. In case of a contamination of the labcoat, sterilize the labcoat first, with bleach or by autoclaving, before washing.
4. Clean and sterilize spills immediately. Report serious contamination immediately to the BVF.
5. It is absolutely prohibited to eat, drink or smoke, or to have cups, plates, mugs or silverware in the VMT area.
6. Pipetting by mouth is prohibited. Used pipettes are collected in a disinfecting solution.
7. Prevent aerosols. These may be created by splashing drops, pouring of liquids, discharging pipettes, opening of moist plugs, using inoculating loops that are too hot. Use needles only if there is absolutely no alternative.
8. Glassware and instruments that have been in contact with GMO's (Genetically Modified Organisms, GGO’s in Dutch) have to be sterilized or disinfected before being washed, reused or discarded. Biological waste has to be collected in autoclavable plastic bags, which are autoclaved before discarding (use indicator tape to demonstrate that the bag was autoclaved).
9. Wash hands with soap and water after work and before leaving the room. Bench surface areas have to be cleaned and disinfected daily. Keep area clean and organized.
10. Record the general nature of the work clearly in a lab journal.

More to consider
Ethidium bromide is a carcinogenic compound that we used when working with agarose gels. To ensure our safety, we created a specific ethidium bromide region in our lab; it is clearly indicated in this area that it is obligatory to use appropriate gloves and keep all the contaminated equipment in the EthBr area. The toxic waste is disposed of in the appropriated manner according the rule and regulations uphold in the laboratories of the university.

Microorganisms involved
We worked with rotting meat, a versatile environment for the growth of Pseudomonas, Salmonella, E. coli and other harmful bacteria. In order to work as safely as possible, we performed our rotten meat experiments in closed bottles and used flow cabinets and procedure masks when taking meat samples. All of the meat was assumed to be biological waste, so it was autoclaved prior to disposal in the appropriate biological waste container. You can read more about food safety in its dedicated page.
Bacillus subtilis. For proof of principle we used B. subtilis strain 168, as it was a safe strain widely used in labs. For our final product we want to use a food grade B. subtilis strain as Bacillus subtilis natto. considered as safe as Lactococcus lactis in yoghurt.
Escherichia coli strain DH5α - a standard laboratory bacterium, widely used as chassis for synthetic biology. This bacterium is considered moderately safe, however, it may cause infections and therefore has to be treated carefully.

BioBricks
We did not find urgent safety issues with our biobricks. The promoters were taken from the genome of B.subtilis 168 and are natural gene parts. Furthermore the other parts of our biobricks are taken from the biobricks in the registry. The Lycopene pigment is considerd food grade and thus safe. GFP and the chromoproteins used as reporters in some of our constructs are widely used reporters that are considerd low risk, however we do not plan to use them in our final Food Warden system and they are only used under safe conditions in the lab. Furthermore we are planning to make our final Food Warden construct without an antibiotic resistance marker.
All our biobricks contain simple modifications that have low or no risk at all. So under normal working conditions in the lab, these biobricks should not pose any danger to the researcher.

Biosafety group
Every research facility has staff responsible for the safety of the labs where people work with GMOs. The University of Groningen is no exception. There are four biological safety officers each charged with a different aspects of safety (site manager, microorganisms, ecology and plants, animal safety) We invited Dr. Juke Lolkema, the safety officer in charge the safety in working with microorganisms, to give a lecture about GMOs and safety in the lab. Dr. Lolkema is responsible for the maintenance of safe microbial techniques in the whole life science building (Linneausborg)of RUG. In this lecture, we were given information about rules and regulation of working with GMOs and how to work safely. During this lecture we asked about the safety aspects of our project. He found no apparent safety issues, due to the nature of our GM insert and the strain of B. subtilis we planned to use. However, he raised an issue we needed to address: how do we kill the bacteria after use? We tried to come up with ideas to address this problem and found a good solution: an internal kill switch triggered by the a strong promotor of, for instance, a stress factor. More detail on this solution are found on the evironmental safety page.
After the lecture Dr. Lolkema made an inspection of our lab. No possible safety problems were found.
The safety lecture by an expert provided us with a good understanding into the safety of working in a laboratory. By working in accordance with the safety rules and the use of good commen sense, we provided a safe working environment for the researchers on the 2012 Groningen iGEM team.