Team:Thessaly/Safety
Biosafety
Synthetic Biology and related fields aspire to commercialize genetically enhanced organisms and their products as well, and make them largely available to the public, with the purpose of providing alternative and novel solutions to various challenges (Wright et al., 2013) . In the realm of a bio-based economy, synthetic bacteria emigrate from the narrow laboratory confinements to the world and are used to serve the goals of society, like bioremediation or as renewable, or for therapeutic purposes, like the ones described in the frame of our project (Amalthea). Nevertheless, the issue of biocontainment is of paramount importance. Biosafety strategies need to be employed to ensure the safety of the final recipients of SynBio products, as well as prevent any unintended consequences that may endanger the natural balances of the ecosystem. Working with genetically modified microorganisms (GMOs) requires precautions that can guarantee the safety of humans and the environment, including laboratory personnel, patients treated with GMOs, and other people who could be exposed to these microorganisms (Kimman et al., 2008) .
As iGEM Thessaly team, we take public concerns regarding biosafety very seriously. So, we have made it a priority to guarantee the safety of all stakeholders involved, especially the safe consumability of our product. For this purpose, we have been conducting our research as safely and securely as possible utilizing all possible personal protection equipment, decontamination equipment (autoclaves), and waste discharge disposal management, in compliance with the guidelines and health protocols framing synthetic biology products and of course international standards. In that context, we have integrated multilayer biosafety and biocontainment levels into our final product, not only to ensure that it can be consumed by the patient without any risk or fear but also to protect the dynamics of the natural habitant ecosystem from artificial forms of life. Those strategies are outlined below.
In the stage of product marketing, we plan to use a well characterized non-pathogenic and commercialized probiotic strain of minimal hazard risk to serve as a chassis for our system. That strain is Escherichia Coli Nissle 1917. which is often recommended for alleviating the symptoms of many gastrointestinal disorders, including diarrhea, uncomplicated diverticular disease, and Ulcerative colitis (Scaldaferri et al., 2016). In addition, this biologically enhanced safe strain will be placed inside a capsule that acts as a physical barrier itself, thus preventing any direct contact of the synthetic organism with the host. For the purposes of designing our product in the laboratory we used only risk group level 1 E. coli derivative strains, which pose minimal individual or community risk.
Figure 1. All experimental procedures were executed following high laboratory standards and best practices. Biological safety cabinets were used for medium preparation and cell handling, and autoclaves were frequently used for sterilization and decontamination processes. Top right: Biological safety cabinet, top left: laboratory bench, bottom right: autoclave, bottom left: autoclave.
Moreover, as part of a holistic approach to minimize potential safety risks, more targeted and robust approaches need to be implemented, so as to reduce risks of horizontal gene transfer. Plenty of biocontainment methods are described in literature, such as using metabolic auxotrophs or integrating toxin-antitoxin circuits inside the chassis.
Kill switches constitute genetic circuits, that, upon triggering, lead to the bacterias’ imminent death, hence preventing escape to the host or the external environment. These multiple layers of biocontainment can ensure that the final commercialized product is safe to use and consume.
The mechanistic framework of TA modules is quite simple. A cell containing a “toxin-antitoxin” plasmid, expresses a labile and unstable “anti-toxin” molecule, which in turn neutralizes the more stable “toxin” molecule. In plasmid-free daughter cells, the “antitoxin” molecule is rapidly depleted, due to having a low biological half-time horizon, thus leaving them susceptible to the toxic activity of the “toxin” molecule inherited from the mother cell. This mechanism effectively reduces the proliferation of plasmid-free cells in growing bacterial populations (Stieber et al., 2008). After intragroup and intergroup discussion with the iGEM team from the Ohio State University, and in line with appropriate biosafety guidelines and standards, we have concluded that a two-tier module, utilizing auxotroph bacteria in conjunction with “kill switch” genetic incorporation, would present a viable solution to suppress biosafety concerns.
Our proposed “kill switch” circuit is based on the mazEF toxin-antitoxin module. It is considered to be an “addiction module”, composed of two genes, mazE & mazF. The first gene, called mazF, encodes a protein that is long-lived and toxic, while the second gene that produces mazE protein is short-lived and antagonizes the toxic effect. Hence, the term “addiction module”, for the bacterium cannot survive without the protective capacity of the antitoxin counterpart, they are practically “addicted” to it. Normally this module enables programmed cell death in E. coli bacteria under conditions of cell stress, particularly starvation in terms of amino acids or carbon depletion around the bacterias’ microenvironment (Aizenman et al., 1996).
Biosafety guidelines compel us to take any appropriate measures necessary to contain the genetically engineered entities, in case there is either escape to the environment or undesired colonialization inside the human body. For these purposes, we have placed the mazF toxin gene under the control of two inducible promoters, the ParaBAD, an arabinose-induced promoter, and a cold-induced one stemming from the cold shock CspA gene. To avoid the basal expression of the lethal mazF, the mazE gene producing the antitoxin product will be put under the control of a constitutive promoter, inactivating any leakage mazF product.
In case the capsule breaks resulting in the bacteria being released inside the patient’s body, exogenous consumption of arabinose shall trigger the activation of the mazF gene, leading to the bacterias’ imminent death. When the capsule will be excreted by defecation from the human body in the stools, the temperature difference will turn the switch on of the cold-induced promoter upstream of the mazF toxin, thus killing the bacteria and safeguarding the environment form the escaping of recombinant microorganisms.
Furthermore, a fruitful partnership with the IGEM Ohio State university resulted in some interesting alternatives. The iGEM Ohio State university team suggested that we knock out the alaR and IlvE genes from the bacterias’ genome and adding L-alanine, L-valine, and leucine inside the capsule for the cells to survive, creating, therefore, a metabolic auxotroph.
Last but not least, apart from biosafety concerns, it was of vital importance to ensure data protection and data privacy from the patients we communicated with during this project. Establishing channels of communication with stakeholders was a core aspect of our project. Nevertheless, our communication with each other and with IBD patients was restricted, making our goal even more difficult to reach. But our determination to work with them and be able to see the problems they have to face, through their own eyes, was greater than any obstacle. Due to the world pandemic of SARS-CoV-2, this initiative was not possible to happen face to face. So, we designed questionnaires that were distributed to the patients, with the help of Dr. Helen Papadimitriou, an IBD specialist from the General Hospital of Larissa. The questionnaires were about the difficulties that they may experience in their diagnosis and treatment, and how they believe that our project could help them.
First, we read iGEM’s policy on Human Subjects Research very carefully. For formal permission, we discussed it with our university professors/PIs. The questions that we provided on the questionnaires were based on International Human Subject Research Resources and especially with DIRECTIVE 95/46 / EC OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 24 October 1995 for the protection of individuals with regard to the processing of personal data nature and the free movement of such data. Contributing to the survey was voluntary and they could suspend its completion or withdraw at any time. Any personal data will remain confidential. Therefore, we adhered to all legal formalities.
In a nutshell, to the best of our knowledge, all safety and data protection parameters have been fulfilled.
References
Aizenman, E., Engelberg-Kulka, H., & Glaser, G. (1996). An Escherichia coli chromosomal “addiction module” regulated by 3′,5′-bispyrophosphate: A model for programmed bacterial cell death. Proceedings of the National Academy of Sciences of the United States of America, 93(12), 6059–6063. https://doi.org/10.1073/pnas.93.12.6059
Kimman, T. G., Smit, E., & Klein, M. R. (2008). Evidence-based biosafety: A review of the principles and effectiveness of microbiological containment measures. Clinical Microbiology Reviews, 21(3), 403–425. https://doi.org/10.1128/CMR.00014-08
Scaldaferri, F., Gerardi, V., Mangiola, F., Lopetuso, L. R., Pizzoferrato, M., Petito, V., Papa, A., Stojanovic, J., Poscia, A., Cammarota, G., & Gasbarrini, A. (2016). Role and mechanisms of action of Escherichia coli nissle 1917 in the maintenance of remission in ulcerative colitis patients: an update. World Journal of Gastroenterology, 22(24), 5505–5511. https://doi.org/10.3748/wjg.v22.i24.5505
Stieber, D., Gabant, P., & Szpirer, C. Y. (2008). The art of selective killing: Plasmid toxin/antitoxin systems and their technological applications. BioTechniques, 45(3), 344–346. https://doi.org/10.2144/000112955
Wright, O., Stan, G. B., & Ellis, T. (2013). Building-in biosafety for synthetic biology. Microbiology (United Kingdom), 159(PART7), 1221–1235. https://doi.org/10.1099/mic.0.066308-0
