Team:TU Darmstadt/Safety
Safety
On the use of GMOs
As with most projects in the field of synthetic biology our project heavily relies on methods of genetic engineering. Altering an organism beyond the extent of natural recombination always involves careful evaluation of biosafety and biocontainment issues. Any genetic modification undertaken may carry both great potential benefits, as well as risks to humans and the environment. It must therefore be examined for its use, necessity, and safety prior to laboratory introduction. Please also find below our discussion on the issues related to dual use. In our project we plan on utilizing the bacterial strains Escherichia coli BL21 and Top10 for early laboratory studies and Bacillus subtilis GP1672 as final application in a biofilm for sewage purification in waste water treatment plants. Both microbial strains have been extensively studied in literature and find wide application as laboratory strains [2,3]. They are both listed on the iGEM white list of organisms not requiring additional check-ins. All experiments are planned to be carried out in an S1 laboratory as no organism or part used exceeds risk group 1.How do we genetically modify our Microorganism?
Our main focus is to utilize the degradational properties of the enzymes CueO/CotA (laccase enzyme family) and EreB (erythromycin esterase type II) for the purification of sewage. In our application, the enzymes are displayed in the bacterial biofilm matrix via fusion to the matrix protein TasA. Therefore, the bacteria have to be modified to express the corresponding fusion proteins. With the exception of CotA – which is native to B. subtilis - the other genes represent heterologous genes that have to be introduced. Moreover, a quorum sensing based kill switch is introduced via plasmids for demonstrational purposes and as a genomic integration for its final application. The exact components of the latter are elaborated in our kill switch section.Is genetic Engineering necessary?
To our knowledge, no microbial species is currently capable of degrading azithromycin or erythromycin other than various pathogenic strains of E. coli from which the ereB gene was first isolated [4]. We evaluate the cultivation of pathogenic bacteria with antibiotic resistance genes in the context of iGEM as reckless and highly hazardous. Our requirements for the bacterial strain are that is must be non-pathogenic as well as capable of biofilm formation. Because no such strain simultaneously possesses the ability for macrolide degradation, we instead resort to genetic engineering of the well-known risk group 1 strain of B. subtilis GP1672. One of our main concerns in working with artificial antibiotic resistance genes is avoiding horizontal gene transfer to pathogenic MO, for instance in the case of bacteria leaving our biofilm. By using genetic modification, we gain more control over the prevention of potential antibiotic resistances in pathogen bacteria that might occur through wastewater pollution with pharmaceuticals. Genetic engineering allows for the implementation of a quorum sensing based kill switch, which minimizes the risk of horizontal gene transfer. An extensive explanation of our kill switchs functionality can be found on our kill switch project page.Does the Benefit of our Project outweigh the Risk of antibiotic Resistance being released into the Environment?
One of the antimicrobials we intend to degrade in wastewater is azithromycin. Erythromycin esterase II (EreB) shows sparse substrate promiscuity for azithromycin. We intend to improve EreB activity towards this substance using site directed mutagenesis methods in combination with the results of our modeling group. However, in case of success we create an artificially enhancend antibiotic resistance towards a group of antibiotics that currently top the World Health Organization's list of Critically Important Antimicrobials. If said gene is not properly contained, we create the resistance we are trying to avoid in the first place. We are aware of these risks and counter them by tightly controlling our biofilm with a novel kill switch system. Moreover, the bacteria we would use are not naturally pathogenic. However, due to the significant, ongoing pollution with azithromycin in sewage, potential pathogenic bacteria are continuously exposed to this substance in the wastewater, thus they might develop a resistance over time. Because azithromycin is of such high importance in human medicine, we evaluate that counteracting the current situation outweighs the risks associated with the containment of our biofilm.How does our Project match with the iGEM Safety Policy?
In iGEM, it is generally recognized that some scientific and engineering activities contain the risk of generating or reinforcing resistance to important medical countermeasures, such as antimicrobic resistance. Since a substantial amount of dominantly utilized selection markers are simultaneously listed on the World Health Organization's list of Critically Important Antimicrobials, most projects in synthetic biology are at some point confronted with this dilemma. Whenever possible, iGEM encourages minimizing the use of resistance factors for antimicrobials of critical importance for public health. Our team fully recognizes and supports this policy, and shares the assessment that conducting important science must always include managing associated risks properly. By implementing an artificial antibiotic resistance, we consciously run the risk of environmental release, however we proactively manage this risk using additional safety measures, such as our quorum sensing based kill switch. We intend to substantially decrease the long-term threat of antibiotic resistances that might develop due to untreated wastewater pollution. The risk associated to our biofilm is not merely tolerated, but has been a substantial consideration within our project from the start. Even though laboratory work has not been possible this year, we completed a representative iGEM safety check-in form for the use of our enzymes as part of our considerations on advantages and risks associated with our project, as would be necessary for any planned experiment implementing an antimicrobial resistance not previously conferred to the respective host organism. For considerations on possible misuse by third parties, please see our text below on dual use.What additional Measures are considered for the Containment of the GMO?
In addition to the safety measures described above, at least one of the following systems can and should be installed to disinfect the wastewater and to implement an additional security layer, such as physical filter systems, UV radiation, ozonolysis or chlorine. However, these measures are expensive. The communities which operate the plants therefore might not want to invest into these novel systems because the treatment plants would run at significantly higher operating costs. In practical use of our biofilm, employees of wastewater treatment plants must be trained in dealing with genetically modified organisms as an essential measure of biocontainment. Please consider reading our safety instructions. In Germany, there is a chance that there is no fourth layer of protection and future research would be needed to implement such a layer. This could possibly be achieved by implementing another dependency for the bacterium to survive inside the cell. A conceivable approach would be a regulation of essential genes or external regulation through using UV exposure or chlorine.
What are the Risks of dual use?
A prevalent issue of any project in genetic engineering and microbiology is the potential of dual use. Dual use is best descripted as the potential for any technology, medicine, or in our case, genetically modified organism, to be used for both beneficial as well as harmful applications. Research conducted for the good of humanity may, in the wrong hands, have severe negative consequences. In special regard to biological weaponry, this issue is extraordinarily crucial in the field of genetic engineering. With genetic modification becoming progressively easier to perform, even to laymen, little knowledge is required to put malicious intentions into practice. Because of this, we consider it essential to be fully transparent with the potential misuse of any part we do or would contribute as part of our iGEM project. The enzymes used hold the possibility of degrading at least a few antibiotics that are currently listed on World Health Organization's list of Critically Important Antimicrobials as critically important. Their high level of occurrence in wastewater is not due to chance but due to the high importance in therapeutic application. Should the hypothetical laboratory work of our project be successful, we create a gene sequence that could, if so intended, be abused to reduce or eliminate the sensitivity of pathogenic bacteria usually treated with said antibiotic through simple genomic integration. With little effort, a substantial amount of available antibiotics, namely those of the macrolide family, could be made ineffective, should the antibiotic resistance prevail in the environment. In that case, the pathogenic bacteria might pose a substantial health threat as the primary treatment of choice would be made ineffective. Even if we managed to uphold any safety standard required to contain the gene within our application, the same cannot be said for sequences that were implemented through a third party because of ignorance or with malicious intent.Biosafety Video
Our Team made a short introductionary video for the livestream we did with iGEM TU Kaiserslautern, where we explain biosafty and our novel kills switch. We would like to encourage you to watch it to get an overview over our safety approaches.Safety in the Lab
Before our laboratories were closed due to Covid-19 in March, a day long instruction course and safety training for every member of the team had been planned to be carried out by our primary principal Investigator (PI) and student instructors. This would have been prior to us working in the laboratory, introducing us to the equipment and potentially dangerous chemicals we might work with, as well as how to behave in the lab, what to wear and how to regard to hazardous situations. If we had worked in the lab, one experienced team member would have always been paired with a rather unexperienced member to support them in the first phase. We thankfully got the opportunity to perform the flow chamber experiment in the laboratory. Due to safety precautions one team member was allowed in the laboratory and got an individual safety instruction. The experiment was performed in the laboratory of Johannes Kabisch at TU Darmstadt, which is biosafety level 1 (S1). This means no pathogenic organisms were used. While being in the laboratory, lab coat and gloves were worn as protective gear as well as safety goggles for more dangerous materials. All safety regulations required by German law were followed. Any gear that came into contact with microorganisms was sterilized before disposal to prevent any bacteria from escaping the lab. Additionally, everybody was required to wear protective masks and keep their distance from each other as is advised during the COVID-19 pandemic.Safety Form for the use of B-TOX
While working with genetically modified organisms holds lots of opportunities, it also brings some risks. Since our biofilm is a modular system and can easily be adapted to other conditions and environmental influences by exchanging the immobilized enzymes, the risk of dual use also threatens our project. Another point is to make sure that our biofilm is being handled responsibly and safe. This means that the organisms will not be released and containment is ensured. It is important to us to prevent any misuse of B-TOX. As one of our experts, Sybille Gaisser suggested we wanted to come up with a way of checking on who orders our system. For that, we have developed a safety form that has to be filled out if people were interested in using our biofilm.
References
[1] COUNCIL. (21. May 1991). Eur-Lex. https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:31991L0271&from=EN, accessed on 25.10.2020 [2] Errington, J., & Wu, L. J. (2017). Cell cycle machinery in Bacillus subtilis. In Prokaryotic Cytoskeletons (pp. 67-101). Springer, Cham [3] Cui, W., Han, L., Suo, F., Liu, Z., Zhou, L., & Zhou, Z. (2018). Exploitation of Bacillus subtilis as a robust workhorse for production of heterologous proteins and beyond. World Journal of Microbiology and Biotechnology, 34(10), 145 [4] Morar, M., Pengelly, K., Koteva, K., & Wright, G. D. (2012). Mechanism and diversity of the erythromycin esterase family of enzymes. Biochemistry, 51(8), 1740-1751