Safety regulations in the Netherlands

Last years team of the University of Groningen had a nice overview of the safety regulations in the Netherlands, EU and worldwide concerning GMOs on their safety page.

There are different levels of safety when working with GMOs, these are named ML1-4 and the higher the number the more dangerous the GMOs are. So ML-1 is for organisms which are non-pathogenic and ML-4 is for organisms which are higly pathogenic. ML-1 organisms meet at least one of the following criteria: It does not belong to a species which is known to contain pathogenic organisms; It has a long history of safe use under normal circumstances; It may be a member of a species which can be pathogenic when the genetic material responsible for the virulence is not present; Its non-pathogenicity and non-virulence was proven by adequate tests. ML-2 organisms can cause a disease in man, plants or animals but is unlikely to spread through the population and an effective cure or controlling method is available. ML-3 organisms can cause a serious disease which are likely to spread under the population, but an effective cure or controlling method is available. ML-4 organisms can cause a severe disease which is likely to spread through the population and no effective cure or controlling method is available. Consequently the rules for labwork are different for each classification level. Appendix 4 of the Dutch regulation for GMOs deals with the specific rules to which laboratories working with the different classifications should comply. Students which have worked in a biotechnology laboratory are familiar with ML-1 rules, like cleaning your bench with ethanol, autoclaves and keeping GMO waste seperately. ML-2 labs are slightly stricter than ML-1. ML-3 labs have a lock chamber and ventilationsystem with filters. ML-4 labs have a lock chamber with a shower, special labclothes and more very strict rules.

References: Ministry of VROM on GMOs Criteria for ML-1 bacteria Classification of bacteria (Dutch) Ministry of VROM on biotechnology (Dutch)

Safety in our project

In appendix 1 of the Dutch regulation for GMOs all non-pathogenic organisms are listed. The organisms on the list are classified as ML-1 when an approved vector and a non-pathogenic insert are used. Examples of pathogenic inserts are genes encoding for toxins, pathogenic factors or oncogens. Bacillus subtilis (the organism we work with) and Streptomyces coelicolor (the host were we get our genes from) are both on the appendix and therefore considered safe and ML-1. So we do not expect dangers for either the researchers, the public or the environment. Ofcourse we did think about this a bit more in the sections found below.

Safety at the university

Within the University of Groningen the organization of biological safety, including all activities that are carried out with genetically modified organisms (GMOs), is the legal responsibility of the Board of the University of Groningen. The Faculty Boards have been mandated by the Board of the University to implement the rules as set out in the GMO Regulations within their own faculty. Biological safety is ensured on the central level by a biological safety organization consisting of the biological safety officers (BSO) as well as a staff member of the Health, Safety and Environment Service (HSE). The HSE coordinates notifications, maintains the register and, in cooperation with the BSO, advises the Faculty Boards and the University Board in the field of biological safety. The central organization is complemented on the local level by responsible officers. BSO officers are members of the staff office and can report directly both to the University board and their own faculty.

For each project a responsible officer will be appointed by the University Board on the recommendation of the Faculty Board. This officer is responsible for various aspects, including day-to-day matters concerning the activities involving genetically modified organisms. We consulted the local safety officer (Juke Lolkema), responsible for our iGEM project and asked him what his thougts about our project were.

For every action taken with GMOs, permission is needed. So for every project permission has to be requested via official forms. The building itself also has to be suitable for GMO experiments. iGEM is a student project and therefore rules for practical courses apply and our supervisors are responsible for the safety issues. Our biology building only has ML-1 and 2 laboratories. Our iGEM project is classified as ML-1, which means that normal rules for working with microorganisms apply. This includes working neatly, washing hands after working with GMO and keeping GMO waste separately so it can be autoclaved.

ML-1 means that our organism (Bacillus subtilis) is non pathogenic and that the GMO we create is non pathogenic as well. Lolkema: “You could eat Bacillus if you want, so it is a really harmless bacteria!” To know if your GMO will be pathogenic or not, it is very important to know what function the DNA that you put in your organism has. It is allowed to insert DNA from a pathogenic organism, as long as the sequence you insert does not encode for pathogenic compounds. If you do not know the sequence and function of the DNA you insert in your host, you automatically work at ML-2 level, which has slightly stricter regulations. The main difference is that the door of ML-2 labs has to be locked when nobody is present, that you have to be even more careful and neat and that you have to fill in a labbook at the entrance of the lab to state your activities of that day. In an ML-1 lab a personal labbook in which you keep track of your experiments is enough.

Everybody in the iGEM team needs to know what is in the lab permit for the project. It is the responsibility of the supervisor that the students know this. Since our supervisor works a lot with Bacillus subtilis, a permit for working with this organism is easy to obtain. Another permit has to be requested to work with the chaplin genes from Streptomyces coelicolor and for the vectors used to insert the genes into Bacillus subtilis. Both organisms are on the appendix of the Dutch regulation GMO, so they are both considered safe and ML-1. Lolkema: “When working with GMOs, one has to keep track of his or her experiments in a notebook. There has to be a table of contents at the beginning and a list of the GMO created at the end. In between the experiments should be described. This is necessary because an inspector has to be able to see what happens in a lab in an easy way.”

In our project, we want to use our bacterium Bacillus subtilis to coat the surface for us. So when we want to coat the hull of a ship for example, living GMOs would be released outside the lab. We were thinking to build a kill switch into the bacteria which would let the bacteria die after they formed a biofilm (the coating). Lolkema: “A kill switch would solve your problems, because it is not allowed to bring living GMOs outside the lab, but genes of dead organisms are not a problem. When you would take your project into production, you would have to go through several permit steps. First you need a permit to grow cultures larger than 10 Liters. Then you would need to do some field experiments which also need a special permit.” Since legislation only deals with living GMOs, we think that it would be easiest to make an ML-1 lab were the hull of the ship could be coated and than take the ship out of the lab after the cells have formed the coating and died due to our killswitch. This way there will be no living GMOs released in the environment during the coating process.

Biobrick Safety

Our biobricks do not pose any health risks or toxicity in the environment, however care should be taken when mass producing the chaplins. Chaplin proteins form very persistent amyloidfibers that are highly surface active. In their natural environment they are already occurring in without causing ecological damage. Because our biofilms would be grown inside an protected area with a build in kill switch dispersing of the bacteria would be minimized.

Amyloid fibres have been associated with many neurodegenerative diseases, like Alzheimer's or Huntington's disease. However, Chaplins fall under the category ‘non-disease and functional amyloids’, like the Curli E. coli Protein (curlin), and pose little risk to public safety. Rather they could provide information on the formation and degradation of these type of fibers and thereby helping in understanding more about the behavior of the amyloid fibers, because of the similarities in structure.

Future ideas for biosafety

We thought about safety issues and how it is possible to improve the safety and use of BioBricks by adding a section safety notes in our standard, which is required, even if no additional safety measurements are needed (BBF RFC 52) In this way, researchers are required to think about the safety when delivering the part to the partsregistry.