Biosafety
Biosafety is an important part of developing genetically modified organisms (GMO). Teams participating on iGEM have to complete and sign extensive safety reports to describe the nature of their work, and how the teams will prevent their project to harm people and the environment.
Upon recognising the environmental and energy challenges posed by conventional solar panels, (see our description page), our team embarked on a journey to harness the power of genetically engineered cyanobacteria in solar energy production. Throughout our project's evolution, we've remained dedicated to addressing end-users needs, such as consumers, solar panel installers, and manufacturers. We wanted to create a product that offers enhanced efficiency and meets safety standards. This commitment to safety has guided us in navigating the complexities of using genetically modified organisms in real-world applications. Crucially, these achievements were made possible through continuous dialogue and collaboration with key stakeholders, including scientists, engineers, and environmental experts in the field.
Our project is the development of a Bio-photovoltaic cell using Synechocystis sp. PCC 6803 expressing cytochromes of the MtrCAB complex and CymA, from Shewanella oneidensis MR-1 to improve extracellular electron transfer (EET), making the cells more suitable to conduct electricity. In addition to the production of electricity, our aim was to make our cyanobacteria solar panel more sustainable since one of the main issues with traditional PVs is that they are not easily recyclable. Our cells besides expressing MtrCAB and CymA to improve conductivity also express higher levels of two amino acids, histidine and lysine. Cyanobacteria already showed to have similar composition to pollen except for their composition of lysine and histidine (the levels are lower). We also wrote a detailed report on the GMO regulations our panel would go through in case we would attempt to release it to the market.
Our Solar Panel and Regulation
We have proactively addressed regulatory concerns from the early stages of our cyanobacterial solar panel project. Recognising that our product would ultimately be applied in the field, we initiated efforts to understand and comply with the regulatory frameworks in the US, UK, and EU. Our commitment to safety and responsible innovation led us to develop a comprehensive strategy that includes a kill-switch mechanism incorporating toxin-antitoxin expression and a layered containment system (see our project description page).
These containment systems mitigate concerns related to GMO regulation. In the European Union (EU), pertinent directives encompass the deliberate release directive (Directive 2001/18/EC) and the contained use directive (Directive 2009/41/EC). Our GMO solar panel does not align with any of these directives and could be classified as 'contained release' case, where GMOs are outside of standard research and industrial facilities but contained by genetic and physical strategies. Projects like ours that do not fit easily into current regulatory frameworks can struggle to get approval despite considerable efforts to ensure safety of the product. In the Biosafety Report, we discuss how current EU GMO regulations could be adapted to support innovative GMO projects and applications.
Our project introduces a unique element where the genetically engineered cyanobacteria, once no longer in use for the solar panels, are intentionally rendered non-viable (dead) before being repurposed as a feed source for bees. This transformation prompts additional considerations within the regulatory landscape. It is essential to evaluate any specific regulations related to the use of GMO-derived materials as bee feed, as this aspect may fall under different regulatory frameworks than those governing 'contained release'. This assessment will be conducted in collaboration with relevant regulatory bodies to ensure compliance.
One pivotal moment in our regulatory journey was our meeting with Dr. Lalitha Sundaram (Research Associate in Biological Risk at the University of Cambridge), a regulatory affairs expert with a wealth of experience in genetic technologies. During this meeting, we presented our project, highlighting the safety features we had integrated. Our toxin-antitoxin expression system and containment measures were key topics of discussion. The meeting delved into several critical regulatory challenges, notably the need for a new regulatory category to accommodate our unique project since our solar panel did not fit into any of the current regulatory directives (deliberate release directive (Directive 2001/18/EC) and the contained used directive (Directive 2009/41/EC). Drawing from insights garnered from previous projects, we learned about the potential hurdles and solutions in the regulatory landscape. In particular, discussions revolved around the possibility of gene integration, the necessity of field trials for validation, and how our cells should not have antibiotic resistance genes to facilitate regulatory approval. Our conversation with Lalitha extended to a global perspective, encompassing diverse regulatory approaches in the UK, Europe, and the US. We gained valuable insights into the intricacies of each region's regulatory environment and how they might impact technology accessibility. Lalitha shared her expertise on recent developments in the UK, such as evolving genetic technology regulations and the concept of regulatory sandboxes for experimental projects. The concept of experimental sandboxes, as introduced in the HM Government's 'Pro-innovation Regulation of Technologies Review Life Sciences' report (1), offers a valuable approach. These sandboxes can be likened to a dynamic testing arena, where innovators and entrepreneurs have more flexibility on regulation.
Developing our biocontainment strategy
When we were thinking of our solar panel, the first thing that came to our mind was how we were going to contain our cells. This also showed to be people’s main worry when we talked about our panel during our human practices. When discussing possibilities for the containment system, it was obvious to us that we needed to have a containment system for our cells that would kill them in case of escape to not spread GMO into the environment. This was essential since our cells would be in a panel outside and would also make approval from governmental entities much easier.
When we started looking at possibilities for our containment system, we also wanted to make it the most sustainable way possible to avoid harsh chemicals that, in many cases, are also toxic to humans and would make the project implementation much harder safety wise. The was two main possibilities based on Sebesta et al's work:
- Active strategy: killing cells by expression of toxic proteins
- Passive strategy: knock out of genes to make cells less fit to live outside our panel
We decided to go with the active strategy and designed a kill switch for Synechocystis based on the constitutive expression of a toxin, NucA nuclease, from a different cyanobacterium Anabaena sp. PCC 7120, together with its inhibitor NuiA, which we put under the control of a zinc-inducible promoter. We chose to use zinc as the inducer because it was previously shown that Synechocystis tolerance to Zn2+ ions is relatively high in comparison to other heavy metals with IC50 (half growth inhibitory concentration) ranging between 8 and 16 µM Zn2+. Moreover, it was also found that Synechocystis had a native promoter regulating the copMRS operon involved in copper response, and that this promoter could also be induced by zinc. In our panel, the media where our cells would be, would be supplemented with zinc2+ ions to induce NuiA antitoxin expression, thus counteracting NucA nuclease effects. In the environment, Zn2+ concentration would usually be too low for efficient NuiA induction, thus NucA would cut cellular DNA and RNA, killing escaped cells.
Limitations of our System
Our system has its limitations, and after meeting with experts involved in policy and regulation and scientists (see our human practices page) we came to the conclusion that this system alone might not be enough to pass regulation tests. Our system might suffer mutations that lead to changes in the behaviour of our kill switch (for example, mutations in the zinc promoter might lead to its inactivation, or mutations in the nuclease could impair its structure inactivating or reducing its toxicity), and zinc in the outside environment would need to be monitored. There is also a problem with testing; when testing for the elimination of our bacteria, it should be noted that available methods for monitoring cell survival, like optical density measurements of bacterial growth and colony-forming unit counting for measuring cell viability, are typically done in carefully controlled experiments. These are not the conditions of the outside world, so these methods may fail to capture mechanisms of escape that may arise in the more complex natural environment. Therefore, these techniques cannot prove the complete eradication of genetically engineered strains, as there is always a possibility that the cells could survive in a dormant state. When meeting with Dr. Lalitha Sundaram from the University of Cambridge for our human practices, we discussed the limitations of our system and concluded that we should have a secondary physical/chemical containment for our cells.
We propose a secondary physical/chemical containment for our solar panel to prevent the escape of GM cyanobacteria in case of breakage or any physical damage to the panel (Figure 3). In our design, any excessive external force would cause the inner layer to break first, and the bactericidal chemical contained in the secondary structure would flow into the solar cell, causing instant cell death, and preventing their escape into the environment. In this system, the nature of the chemical has to be carefully considered; it must be relatively cheap, undergo a separate risk assessment keeping in mind its potential leakage into the environment, and be previously tested/approved to be 100% bactericidal to ensure cell death.
Figure 3. Secondary physical/chemical containment system for the solar panel.
What happens if our bacteria get released in the environment?
If GMOs, like bacteria get released to the environment, there is a probability that they might die out or be outcompeted by native species. Species that are considered model species have been grown continuously in laboratories and may have evolved or acclimated to the favourable environment of the lab where they typically have media much richer than anything found in the environment, they are not exposed to UV light, and where predators and competitors are carefully excluded. Also, engineered cells can be disadvantaged by the metabolic burden. These assumptions, however, cannot be taken for granted and it is essential to ensure the complete safety of our panel.
In some cases GMOs may survive and spread in the environment. Some cells form spores, but even though Synechocystis cannot form spores, our cells may enter a state of low or no growth in order to survive in nutrient limited conditions. This state is very difficult ot study in the lab since common assays to test escape frequency, such as growth curves and colony counting, may not capture this mechanism since the extremely slow growth of the persistent state may appear the same as cell death.
How our GMO might interfere with the natural ecosystem
Escaped GMOs may alter fundamental properties of a ecosystem such as the dominant species in a community and the ecosystem’s physical features as well as nutrient cycling (Mack et al., 2000). Specific products produced by GMOs might be toxic to other organisms. The engineered organisms can become food for other organisms, which may alter the environment unpredictably. Horizontal gene transfer from GMOs to other organisms can also have unpredictable consequences, for instance, it could result in the spread of antibiotic-resistance genes as these genes are often used as selection markers for genetic modification. This is the reason why it is important to ensure that antibiotic-resistance genes are removed from the final engineered strain. This is very important to the entities involved in approving projects like ours; we were advised during our human practices on this since retaining antibiotic-resistance genes in a GMO makes the approval much more likely to be refused.
While the field of synthetic biology keeps growing and evolving with novel GMO products being developed, the importance of biosafety is ever-clear as well as a pressing need for regulators to keep up with these technological advancements. Our project is a good example of GMO products implementing different containment measures to increase safety. However, it is important to note that different containment strategies, like genetic kill switches, auxotrophies, and physical and/or chemical containment systems, can never ensure 100% safety. Thus, negative consequences of GMO release and spread in the environment always have to be seriously considered and the risks thoroughly assessed. The intricate GMO regulatory landscape, mainly rooted in the historical split between contained use and deliberate release in the EU, may hinder the progress of innovative synthetic biology projects that defy conventional classification, as exemplified by case studies. Moreover, the current regulatory processes are complex, lacking clarity and transparency, leading to many uncertainties faced by researchers and companies seeking regulatory approval for innovative projects. There are many ways to approach these problems to better align regulatory frameworks with the rapid advancements in synthetic. Some of the suggested solutions are the establishment of the Engineering Biology Regulatory Network (EBRN) and regulatory sandboxes for testing novel products, as well as cloud-based platforms for efficient information sharing and clear regulatory roadmaps. By adopting the suggested reforms and fostering a regulatory environment that accommodates innovation while ensuring safety and environmental protection, the EU and other countries can position themselves at the forefront of pioneering biotechnological developments and contribute to addressing critical global issues. The potential future success of Team Edinburgh's iGEM project and similar initiatives hinges on the adaptability and responsiveness of regulatory bodies to the evolving landscape of biotechnology.