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To ensure safe working practices throughout the competition, each team member attended a general safety briefing on good laboratory practice and working with genetically modified organisms (GMOs), including storage and disposal.
Firstly, we prevented any release into the environment or harm to health during our project by following safety rules and training people to carry out different laboratory tasks. In addition, the laboratory we work in is specialized in working with Bacillus subtilis, which is a great support when needed.
We attended a workshop on biosafety and biosecurity at the BFH European iGEM Meetup where project-specific risks were assessed and no specific risks were found. It was also estimated that, in general, the project is not a dual-use project. Thus, nobody could use it to do harm.
In order to evaluate the safety aspect and the future use of our project, we consulted various experts and biological safety officers.
Firstly, we talked to our supervisor Prof. Thorsten Mascher, a B. subtilis expert, about the safety aspects of our project. We also met with Dr. Ulrike Scholz and Johannes Kautz, who are responsible for biological safety at our university, to discuss the relevant safety aspects of our project and its future use. We also invited Dr. Udo Mücke, who is responsible for the safety of genetic engineering in the state of Saxony (SMEKUL, Referat 45 - Strahlenschutz, Gentechnik, Chemikalien). Unfortunately, he was unable to attend the discussion due to illness, but he kindly answered remaining questions afterwards.
Our chassis of choice B. subtilis holds the GRAS status which stands for “Generally Recognized As Safe“. For a substance or organism to be GRAS, its safety must be widely known, and experts must agree that scientific data confirm that it is safe for its intended use.
In general, B. subtilis is a widely studied model organism which can be easily genetically modified and is often used for industrial production of various enzymes, e.g. using the efficient expression strain WB800N (Barák 2021, Jeong et al. 2018).
In addition, B. subtilis is assigned to risk group 1 as a donor and recipient organism. Risk group 1 organisms are not expected to pose a risk to human health or the environment.
Bacillus subtilis W168, our laboratory wildtype strain, is auxotrophic for tryptophan due to a deletion of trpC2 and would be unable to survive in a normal environment.
In our project, B. subtilis was genetically modified to harbor our target constructs. Hence, our generated strains fall under the Genetic engineering act (GenTG, Gentechnik Gesetz) which is the central legal basis for dealing with GMOs in Germany.
The production and any use of GMOs (including storage) is genetic engineering work (§3 No. 2 GenTG) which may in principle be carried out in genetic engineering facilities only.
As B. subtilis is a risk group 1 organism and our genetic modifications are not expected to increase the risk potential, the GMOs we produced are also classified as risk group 1. This means that all work with these organisms must be carried out in a S1 (biosafety level 1) laboratory.
A special feature of B. subtilis is its ability to form endospores. An endospore is a dormant stage of the Bacillus life cycle which enables the bacteria to survive harsh environmental conditions. In our project genetically modified spores of B. subtilis W168 were used.
Our spores can be seen as vehicles for DNA and are therefore in principle no GMOs according to §3 No. 3 GenTG. This act states that GMOs are 1) living organisms and 2) capable of transferring their genetic material (actively, e.g. by proliferation). Spores do not meet these criteria as they are dormant, have no active metabolism and cannot actively reproduce. However, the production of our spores and their ability to germinate into a vegetative cell makes them a GMO according to §3 No. 3 GenTG. Therefore, working with them requires a biosafety level 1 facility.
In this regard, Prof. Mascher referred to the LMU Munich iGEM 2012 project, in which B. subtilis was modified to lack the ability to germinate (GerminationSTOP), making spores a safe platform for synthetic biology. This project also evaluated the safety of B. subtilis and its spores in general including a discussion with a biosafety officer.
They showed that none of 2•107 spores were able to germinate with the GerminationSTOP. They also tried to establish a suicide switch for their spores. With these two mechanisms, they added a second and third safety layer, with the first safety layer being Bacillus auxotrophy for tryptophan, making it unable to survive in a normal environment.
In the future, we plan to introduce a similar GerminationSTOP to our spores, preventing them from germinating.
In our project we used cellulases from Bacillus subtilis, Bacillus pumilus, Bacillus halodurans, Paenibacillus polymyxa and Acetivibrio thermocellus. We also used BhrPETase from Bacterium HR29. Gene sequences of these proteins were found in papers and databases and the parts were synthesized by IDT. All constructs used in our project are save according to the Biosecurity Sequence Scanner from Asimov Kernel. This includes parts like promotor or regulators. All plasmids we used are also safe and widely used in the iGEM framework.
All the genes encoding our enzymes can be assigned to safety level 1 as they are not known to pose any risk to human health or the environment and derived from non-pathogenic, non-hazardous organisms.
If the cellulase and PETase genes were somehow transferred to another organism, it is likely that PET and cotton would have to be pre-treated for proper degradation. In addition, the engineered genes originate from organisms that produce these enzymes in nature.
In some of our expert talks we also discussed the up scaling of the project. Both Prof. Mascher and Dr. Werner noted that there could be some challenges, as an industrial application of our project would have to take place in an appropriate genetic engineering (production) facility with safety level 1 (see Endospore as an immobilization platform), making its implementation more complex and expensive.
Industrial use of our spores in an S1 production facility would require prior application by the operator to the responsible state authority (in Saxony: SMEKUL, Dr. Udo Mücke) according to the GenTG. In particular, the security measures in accordance with §§ 13 ff. GenTSV and Annex 2 Section B. I. GenTSV (security measures for the production area for security level 1). (Dr. U. Mücke, personal communication, September 17, 2024)
In principle, (unapproved) GMOs must not be released into the environment. Therefore, production must take place in a closed system (e.g. a fermenter), aerosol formation must be avoided and precautions must be taken to prevent uncontrolled release of the GMOs (collection device in the event of leakage or similar, see appendix 2 B.I.a. 6. - 9. and b. 4. - 9. GenTSV). According to §23 GenTSV, wastewater and waste containing GMOs must be inactivated before disposal. (Dr. U. Mücke, personal communication, September 17, 2024)
If our GMOs were a biological safety measure (§7, §8 GenTSV), disposal would be possible without special pretreatment according to §24(1) No. 3 GenTSV, which would also mean a corresponding reduction in costs. Then, for example, the collection device could also be dispensed with (Appendix 2 B. I. a. 9. GenTSV). (Dr. U. Mücke, personal communication, September 17, 2024)
In Germany, the Central Commission for Biological Safety (ZKBS, Zentrale Kommission für die Biologische Sicherheit) is responsible for the recognition of a GMO as a biological safety measure. The ZKBS is a voluntary expert committee. It advises those people responsible for decision-making in politics and administration by providing technical opinions and thus contributes to safety in genetic engineering.
In our case, the ZKBS does not consider auxotrophy for tryptophan alone to be sufficient for recognition as a biological safety measure. If we want our strain to be recognized as a biological safety measure due to the additional lack of germination of the spores, a fee-based statement from the ZKBS would be required (individual case decision). When submitting the documents, proof of the non-germination of the spores and, if necessary, their regular testing during the process should be presented. According to the ZKBS statement, deactivation and certain security measures could then possibly be dispensed with (see above). (Dr. U. Mücke, personal communication, September 17, 2024)
The industrial use of GMOs is subject to a strict set of regulations aimed at protecting the environment, health and biodiversity. Before being used in industry, GMOs must be fully tested and approved.
Within the EU, the national genetic engineering legislation on GMOs, such as in Germany (GenTG), is based on the so-called System Directive 2009/41/EC which means that similar requirements also apply in other EU countries. (Dr. U. Mücke, personal communication, September 17, 2024)
In Germany, the Genetic Engineering Act (GenTG) and the corresponding ordinances form the legal framework for ensuring the safe use of genetic engineering. The law applies to the release of GMOs, the placing on the market of products containing or consisting of GMOs and carrying out genetic engineering work in genetic engineering facilities. The Genetic Engineering Safety Ordinance (GenTSV) lays down safety measures for the respective type of facility (e.g. laboratory or production facility) and the established safety level of the genetic engineering work (GenTSV, Gentechnik Sicherheitsverordnung).
When moving GMOs to third countries, the Cartagena Protocol (Protocol on Biosafety to the Convention on Biological Diversity) must be observed. It relates to the cross-border movement of GMOs and aims to ensure that a comprehensive risk assessment is carried out before importing GMOs. In the EU the Cartagena Protocol is implemented into valid law by Commission Regulation (EC) No 1946/2003. (Dr. U. Scholz and Dr. U. Mücke, personal communication, September 10 and 17, 2024)