Laws and Guidelines to be Considered in Denmark

The scope of this part of the paper is to draw attention to some of the laws and guidelines, which we have to consider in Denmark, when we are dealing with genetically modified microorganisms (GMM's). Our project is defined as a 'contained use' project, which means that the organisms we are handling are contained from the environment at large. The following laws are based on the ”Bekendtgørelsen om Genteknologi og Arbejdsmiljø” (eng. The Order on Gene-technology and Working Environment) of 2008, which follows the rules laid down by the European Union in 1990 in the ”Directive on the Contained Use of Genetically Modified Micro-organisms”.

Risk-assessment

One of the first, and indeed one of the weightiest points in the directive on GMM safety, is to ensure the public health and the preservation of the environment.

To that end [to avoid adverse effects on human health and the environment which might arise from the contained use of GMM’s], the user shall carry out an assessment of the contained uses as regards the risks to human health and the environment that those contained uses may pose, using as a minimum the elements of assessment and the procedure set out in Annex III, Sections A and B. [1]

It is required of us to make a thorough risk-assessment, so that we may judge if our use of GMM's poses a threat towards the well-being or safety of human beings, animals, plants, or the environment. To help perform this assessment, the UN has laid down a minimum standard of elements required to make an adequate assessment of the potential harm of an accident resulting in the release of the GMM's into the environment. The following is a list of the minimum elements required:

Assessment factors

1. Assessment of potential harmful effects, defined as
a) Disease in humans, animals or plants
b) Harmful effects resulting from inability to cure disease
c) Harmful effects resulting from organisms establishing themselves in nature
d) Harmful effects resulting from the organism, through natural processes, conferring part of its genome, such as heightened resistance, to other organisms in nature
2. Resulting from
a) The host-organism to be modified
b) The parts inserted into or otherwise used to alter the organism
c) The vector
d) The donor-organism
e) The resulting modified organism
3. Characteristics for the organism's activity
4. How potent the potential harmful effects are
5. The likelihood of harmful effects being realized

Based on this risk-assessment it is possible to rank the project according to the risk, ranking from level 1 to 4, in accordance with the procedure giving by the UN. See appendix I for the risk-assessment we made for our project.
Personal Safety
To be allowed to work in a level 1 laboratory, it is required that there at all times is a suitable instructed person present. At level 2, all personnel in the laboratory are required to have been suitable instructed in lab safety and procedure. All access to the lab by non-members of this group or the lab-staff is to be restricted. All members of our team have in the time prior to the work in the laboratory undertaken a lab-safety-course, thus fulfilling the requirements to work in a level 1 lab.

Substitution

Furthermore, it is not legal to work with any host, donor or vector-system, should another, safer, system, containing the same basic features, be available. If it is possible to find a suitable system, compatible with the intended work, that is safer for humans, animals and plants, or the environment at large, it must always substitute the other, more dangerous system. It is in other words prohibited to take unnecessary risks, or use unnecessarily risky setups. Should a possible substitute system be unreasonably difficult or expensive to acquire, then the risks and benefits must be considered, favoring safety above economical issues. As we're working with relatively harmless strains of E. coli (MG1655 and TOP10 strains), it has not been necessary to locate a safer, compatible host, donor or system, but we have nonetheless attempted to locate such systems for wholesomeness, although without luck.

Assessment by Local Bio-safety Group

Laboratory Safety Course

Team SDU-Denmark consists of students from various fields such as biochemistry & molecular biology, chemistry, physics, medicine and philosophy. Not all are used to working in the lab, and so it was necessary to make sure everyone knew about basic safety precautions to be taken when in the lab. Hence our first step in addressing the safety issue, was to have everyone on our team participating in a laboratory safety course (unless already attended) held by Mogens T. Jensen and Marianne Due from the Committee for Students’ Laboratory Safety. This included a small handbook and information on /practice in: good conduct in the lab, use of safety devices, signs used in the lab, emergency cases (fire), elementary first aid, waste, knowledge about the Organisation for Work Environment at SDU and suggestions to further readings.
Safety of our Laboratories
We are using the labs of the Institute of Biochemistry and Molecular Biology (BMB) and the Centre for Fundamental Living Technology (FLinT). Both are security level 1 laboratories, which is what we need for this project, and the bacteria we are working with, since they are non-pathogenic.

The Group

“Arbejdsmiljøgruppen” (eng. The Working Environment Group) is the local bio-safety group associated with the University of Southern Denmark. During an interview with a representative from this group we explained the project, its scope, parts and procedure. The following is a number of questions concerning the safety and security issues relating to our project, and the essence of their replies.

If they perceived an increased risk due to work being performed by relatively inexperienced students

The project is not considered any more dangerous due to the fact that most of the work in the lab is performed by relatively inexperienced students. As long as the lab's safety protocol is followed, and the fact that the risk-assessment of the work safety group put our project firmly on level 1, they believe that there should be little to no risk to lab personnel or the outside environment. As all students participating in the lab has successfully completed the lab safety course provided by The Working Environment Group, they perceived no increased risk.

If they perceived any danger should the bacteria get out of the lab

They perceived no danger to the environment or the well-being of animals, plants or humans should the bacteria be released into the environment. This is due to the extremely fragile nature of the E. coli strain that we are using in our project. Should it somehow find its way outside of the lab, it would die within a very short timespan.

If there exists an emergency safety protocol in case of accident (i.e. unintentional release of GMM's into environment)

The emergency protocol is still a work in progress, but although it is unfinished it should not pose a breach in safety, as we're only working with a level 1 GMM, which due to its extremely fragile nature cannot survive outside of laboratory environment. This coupled with adherence to the standard laboratory safety protocol, should at all times ensure the safety of the environment.

Overall Assessment

We have at all times upheld the laws and regulations imposed upon us by UN and by the Working Environment Group. We have performed a risk-assessment of our project as required by the UN, as well as following the laws regarding to personal safety and to substitution of potentially harmful host and donor organisms. The work safety group has assessed our project to be a class 1 project, as they have perceived no risk associated with our work. They see no apparent way of weaponizing or in any other way using our project for malign purposes. Thus our project should not pose any threat to the security of the world at large. Although most of the genes inserted into our bacteria are harmless, hyperflagellation is in fact something that increases pathogenicity. Furthermore the bacteria we work with are unable to survive and reproduce outside of laboratory conditions. Should it accidentally be released into the wild, it would lose its plasmids within a very short time span and thus return to a non-GMO state. And as the bacteria we have been working with, namely E. coli MG1655, is a naturally occurring bacteria, it should not pose any threat to the environment at all. As long as the normative work safety protocols were followed they could not perceive any danger due to the work being performed by relatively inexperienced students. Thus they perceived neither security nor safety issues with our project.

References

[1]: Article 4.2 of the Directive on the Contained Use of Genetically Modified Micro-organisms, UN, 1990
[2]: http://www.bmwf.gv.at/fileadmin/user_upload/forschung/gentechnik/2009-41-EC.pdf
[3]: https://www.retsinformation.dk/Forms/R0710.aspx?id=121099

Appendix 1

Risk-assessment

Title: Synthesis of hyper-flagellated phototaxic E. coli.

Purpose: To create a bacteria that can induce a micro-flow regulated by light.

Host: Bacteria, E. coli.

Strains: Mg1655 and TOP10.

Donor: Coding regions amplified from naturally occurent organism. Photosensor: N. pharaonis, S. enterica serovar typhimurium. These create a fusion protein. Sr2 + Htr2 from N. pharaonis, Tar/CheW from S. enterica.
Retinal: nina B from cDNA from D. Melanogaster
Flagella: FlhDC from E. Coli

Vectors:
pSB3TS
pSB3CS
pSB3K3
pSB1A2

Inserts:
BBa_K343004
BBa_K343006
BBa_K343007

Risk-assessment

Host:
Bacteria:
E. coli is naturally occurring and the strains used for amplifying vector-DNA/proteins is not reported pathogenic
Al material used in bacterial work is autoclaved and/or inactivated with iodofor

Strains:
A cell-culture from a higher eukaryot which does not contain any endogene vectors that would be able to mobilize parts of the transferred genetic material.
The strains used have not been reported pathogenic
The cellular strain is very fragile and is unable to procreate or survive outside of laboratory conditions, as they need the correct temperature, humidity, pH, CO2, O2 and nourishment
Al material used in bacterial work is autoclaved and/or inactivated with iodofor

Donor:
Naturally occurring healthy genes of insect origin and it is not believed to be able to transform/infect human cells in vitro/vivo. The risk is therefore considered to be minimal. The S.E. gene has homology in E. coli and is therefore not considered to pose any threat.
Vector:
Vectors are of pUC or pOT2 origin and nothing from the vector has human recombinations/infection potential and the risk of working with these strains are therefore believed to be minimal.
Our vector is in addition equipped with resistance to antibiotics and cannot exist without it. Should discard the resistance if not within a antibiotic environment.

Insert:
Is naturally occurring genes with well-defined tags and it is believed that they cannot transform/infect human cells in vitro/vivo. The fusion-protein has had limited testing, but is also considered safe. The risk is therefore believed to be minimal.

Health-aspects of the final GMO:
Bacteria not exposed to antibiotics will discard the plasmids within a very short timespan.
The bacteria are modified with plasmids, and will return to a non-GMO state within a short time-span. The modification is not infective/self-reproductive in humans. It is not believed to pose any threat towards human health.
We have at no point worked with any self-reproductive or pathogenic material

Assessment: Class 1

Appendix II

This is the Danish law on GMOs, called "Bekendtgørelse om godkendelse af udstilling og informationsformidling med genetisk modificerede organismer" (The Law on Approval of Exhibition and Information on Genetically Modified Organismes).

A description of the modified organism

A full description of the modified organism should ideally contain the following information. This is only ideally, as it would be a far too time-consuming procedure to fill out all the following information. The lists are supposed to be mere guidelines.

A. characteristics of the host and donor organisms

1. Name(s) of the organism(s) in question
2. Origin of organism(s) in question
3. Information on the reproductive cycle of the parental organisms as well as the host
4. Description of any previous genetic modification
5. Stability
6. Details concerning pathogenesis, virulence, infectivity or toxicity
7. Characteristics of endogene vectors: a) Sequence b) Mobilization c) Specificity d) The presence of resistance-genes
8. Host spectrum
9. Potentially significant physiological traits and the stability of these traits
10. Natural habitat
11. Significant role in environmental processes
12. Competition or symbiosis with other naturally occurring organisms
13. Ability to create survival structures (i.e. the ability to create spores)

B. characteristics of the genetically modified organism

1. Origin of the genetic material used to modify the organism, as well as the intended functions of this material
2. Description of the modification, including the method of vector insertion in the host organism, as well as the method used to create the genetically modified production-organism
3. The function of the genetic modification
4. Origin and characteristics of the vector
5. Structure and size of vector in the genetically modified production-organism
6. Stability of the organism with respect to genetic traits
7. Mobilization frequency of the inserted vector and/or the organism’s ability to transfer genetic material
8. Activity of the expressed protein

C. Health concerns

1. Toxic or allergenic properties
2. Product risks
3. The genetic modified organism’s pathogenic properties compared with the donor – or the host organisms or possibly the donor organism
4. Colonization ability
5. If the organism is pathogenic to humans, who are immune competent: a) Cause illness and the pathogenic mechanism, including invasiveness and virulence b) Infectivity c) Infective dose d) Host range, possibility of change e) Possibility for survival outside the human host f) The presence of vectors or other distribution areas g) Biological stability h) Resistance patterns against antibiotics i) Allergenicity j) Chance for suitable disease treatment

D. Environmental concerns

1. Factors that might affect the organism’s ability for survival, reproduction and it’s ability to spread in the environment.
2. Techniques for detection, identification and surveillance of the modified organism
3. Techniques for detection of transfer of genetic material to other organisms
4. Known and expected habitats of the modified organism
5. Description of ecosystems into which the organism could spread in the event of an accident
6. Expected result of interaction between the modified organism and naturally occurring bacteria that would be affected in the event of an accident
7. Known and expected effects on animals and plants, with regards to pathogenesis, virulence, infectivity, toxicity, allergenicity, colonization
8. Known or expected contribution to bio-geo-chemic processes
9. Methods for decontamination of the area in the event of an accident