Implementation of our project requires us to apply our project into the real world. As a second phase project, we have worked to evaluate and improve upon our proposed implementation to ensure the best possible outcome for the coral reefs, the marine ecosystems and our communities. The devastating impacts of climate change on the Great Barrier Reef as well as coral reefs around the world have seen a shift in public attitudes towards a greater acceptance of synthetic biology solutions: As we have found in our survey, 75% of our respondents strongly agree/agree with the statement “I support the use of genetic engineering to protect the coral reefs”. Nevertheless, there are numerous concerns that must be addressed when proposing to implement a genetically modified solution into a natural environment. It is important to our team that our proposed implementation takes into careful consideration the technical, safety, ethical and socio-cultural concerns.
During our conversation with Will Howard, we discussed the environmental and ethical concerns that come to light when dealing with GMOs in the environment. He urged us to remember that there have been mistakes made with introduced species in the past which although initially began with good intentions, have had devastating consequences. As a team, we fully recognise the importance of being proactive when considering our responsibilities to the environment as we navigate the implementation of our project. We propose to implement our solution in a three-step phase, starting with the long-term observation of our modified organism in a controlled and contained simulated reef environment. With this approach, our team will be able to ensure the ‘safest’ possible delivery of our project in the best interest of the environment and our community.
Our Proposed End Users
In the development of our project, the UNSW iGEM team has considered who our' proposed end user' should be and how they should implement our solution in the real world. Our proposed end-users are the government and non-government organisations whose interest is protecting and conserving the environment and the reefs.
In the earlier stages, our team considered broadening our horizons and exploring the possibility of commercialisation to receive funding and move forward with our proposed solution. However, upon discussing this possibility with David Burt, the Director of Entrepreneurship at the University of New South Wales, we were given the opportunity to critically evaluate the purpose of our project and how our implementation will align with our values. During this conversation, we asked ourselves the question of "who will be motivated to invest money into our solution?". This question made us consider the motivations we want our proposed end-users to have and whether our end product would be commercially profitable for them. We have since decided that our proposed end-users should not be motivated by profit. Instead, our team proposed that our end user's incentive should be a desire to protect the coral reefs from climate change. Thus, we have decided that our project's goals do not align with the commercialisation route.
Our team believes that our end users should be accountable for the people connected to the Great Barrier Reef. Moreover, our end users should be capable of ensuring that our stakeholder's voices are valued and integrated into the implementation of our solution. Finally, our proposed end users must have the scientific capacity and authority to deploy our solutions for the sole benefit of the coral reefs.
The government is a potential end-user with a duty to listen to and act upon the community's best interests. The Great Barrier Reef Marine Park Authority ('GBRMPA') is a specialised government agency with motivations secured by legislation to act in the best interest of the GBR. The GBRMPA is a well-respected agency that works with various partners, advisers, and stakeholders to manage and conserve the GBR for future generations. They are identified as an ideal end-user for our solution since; as a government agency, they are accountable to the people and the best interests of the community.
Another suitable end-user are non-government organisations (NGOs). The incentives of NGOs align with our intentions of using our solution in a non-profit, open-source manner. Additionally, International NGOs such as the Nature Conservancy, WWF, and UNESCO can affect our solution beyond Australian borders. This global reach would allow us to expand our solution to benefit coral reefs and communities around the world. As an end-user of our solution, NGOs have independence from the government, offering the potential for a more straightforward path of action that is less likely to be swayed by political change.
Implementation of Our Project in the Real World
Ex-situ uptake and In-situ implementation
When considering the proposed implementation of our project, we spoke with David Wachenfeld, Chief Scientist of the GBRMPA. In our discussions, he talked to us about the vast complexities of the GBR, and the many aspects of the marine ecosystem that are still exceedingly unknown. Due to the complex and interconnected nature of the marine environment, it is impossible for us to consider the greater impact of our solution on the coral halobiont and greater reef ecosystem unless we are able to study our product in a simulated marine environment.
Thus, the first stage of our project’s proposed implementation involves the ex-situ testing of our modified Symdbioidnium in a simulated coral reef such as the Australian Institute of Marine Science's National Sea Simulator (SeaSim, which is a world-class marine research aquarium facility in which scientists provide fine control over environmental variables. Our next step would involve the transfer of our modified organism into an in-situ marine environment that has low levels of biodiversity to limit the potential negative impacts on biodiversity. Lastly, if our previous stages are successful, our modified Symbiodinium-coral system can be released into a natural environment with normal biodiversity conditions.
To ensure the safe and responsible implementation of our proposed solution, our team has developed a three-stage approach to mitigate risk to the coral reefs, the surrounding biodiversity and the marine ecosystem.
Long-term observation of modified Symbiodinium impacts on the coral and greater biodiversity, within a controlled and contained environment simulating ocean conditions.
In-Situ Testing In Multiple Areas of Low Biodiversity
Long-term observation of modified Symbiodinium impacts on coral in a natural yet depleted coral environment. This reduces the risk of negative impacts on other coral species and surrounding biodiversity.
In-Situ Release In Area of Normal Biodiversity
If both previous stages are successful, then the genetically modified Symbiodinium-coral system may be released in the natural environment with standard biodiversity conditions. Continual observation of the environment would be necessary.
Another consideration that has arisen since formulating our three-step approach was discussed during our meeting with David Wachenfeld. In our conversation, David talked about the interconnectedness of the coral reefs, and how the spawn from certain reefs, which he referred to as the ‘hub of reefs’ are more likely to end up on other reefs by the action of the ocean currents. This knowledge can be utilised in the final stages of our implementation, as we can select reefs to release our modified Symbiodinium-coral system where our solution will have maximum impact.
Licencing and Permit Applications
The common denominator between all of our stakeholders, in regards to the implementation of production, was the importance of rigorous testing before releasing our genetically engineered algae symbiont in an in-situ environment. Kevin Gale mentioned that we would have to first obtain a licensing permit from the Great Barrier Marine Park and comply with the set standards of the Office of the Gene Technology Regulator (‘OGTR’) standards
Since our application for the former would fall under the bracket of research, the first step is to receive permission from managing agencies, unless it satisfies the requirement of limited impact research by an accredited institution. The application must include relevant information such as justifying the location of choice and the proposed route of research. The applicant must assess the severity of the risks if implemented, and provide additional documentation such as general public consultation, targeted consultation with Traditional Owners, government agencies or subject matter experts. Upon being lodged, is the severity of the risk is assessed by the managing agencies, and the respective actions will be taken (Great Barrier Reef Marine Park Authority, 2017). Although we are only in the early stages of our project, we would hope to aim for the assessment approach that requires a public information package. This would identify our proposed activity as medium risk, which would require our application to have public comments, so as to assess these foreseeable risks.
Genetically modified organisms cannot be dealt with unless it has been approved by the Office of the Gene Technology Regulator. This approval process is dependant on three factors:
- Organisation Accreditation: Applying for a licence requires an organisation to accredit this genetically modified organism.
- Institutional Biosafety Committees (IBCs): This organisation assists with the adherence to the regulatory scheme laws, by either conducting on-site assessment for low-risk options or by reviewing the application of high-risk options, before being passed on the Office of the Gene Technology Regulator.
- Facility Certification: Dealings with certain genetically modified organisms can only be between certified facilities, so this must be pre-approved as well.
The concept of social licensing was explored in the first phase of our project and was reiterated during the conversations with our stakeholders this year. In a broader sense, we would like to continue engaging with our four subgroups of stakeholders - Academics, Businesses, Government Bodies and Traditional Owners, so as to be on the same wavelength and adapt to their changing needs and values.
We would strive to further our human-centred approach by involving more of the public’s perspective regarding our solution. In the later stages of our project, we could hold forums that enable the public to voice their opinions and concerns regarding each phase in our pipeline and hone in on the science communication aspect of our project, which allows us to educate the wider population regarding the potential of synthetic biology to possibly halt the issue of coral bleaching.
A/Prof Daniel Robinson implored us to consider the biocultural protocols during the planning of our proposed implementation framework, which falls under social licensing. Whilst continuously nurturing the relationship with Traditional Owners, it is on the onus of the future UNSW iGEM teams to take into consideration the customary values of the wider community. An example of which is “Acess and Benefit-Sharing”, which describes the methods used to access these genetic resources, and thinking of a long-term plan on how the share the benefits resulted from its use, between those utilising and providing the resources (Convention on Biological Diversity: ABS, 2010).
Biodiversity and the Kill Switch
Considering that our Proposed Solution involved the release of GMOs into the environment, a major environmental concern is the unintended invasion of our modified species outside of its intended environment. If this is not prevented, this may result in changes to the marine ecosystem dynamics and result in loss of biodiversity. In order to limit the potential of this occurring, biosafety considerations must be integrated into our project design that can also be used to inform our experimental approaches. Genetically modified microorganisms can be designed with biocontainment systems such as kill switches or natural/synthetic auxotrophy. These systems will result in cell death upon exposure to certain conditions, such as the presence of a specific molecule or a change in pH. Thus, the integration of our modified Symbiodinium into other species can be prevented by designing a kill switch that is triggered by its expulsion from the coral.
A kill switch for our genetically modified microorganism has been discussed since the beginning of our project development by the previous team. Since existing biocontainment systems for eukaryotic algal symbionts are limited, our team has researched designing a novel biocontainment system specific for Symbiodinium. The novel biocontainment system aims to take advantage of the natural mechanisms by which the Symbiodinium associates with its coral hosts. The interaction of microbe-associated molecular patterns (MAMPs) on Symbiodinium with the pattern recognition receptors (PRRs) on the corals facilitate the endocytosis of this dinoflagellate by the coral tissue (Liu et al., 2018). The microalgae then reside within the coral polyp tissue in a symbiosome vacuole through continued cell surface interaction, separated from the rest of the coral cytoplasm. When expelled from the coral, MAMP/PRR interaction is lost (Davy et al., 2012).
Our novel kill switch will utilise this mechanism to activate a toxin/anti-toxin system. The toxin will be produced constitutively under a weak promoter, which allows the Symbiodinium to survive for a period of time even after its detachment from the coral, allowing a window to introduce the modified Symbioinium to corals during implantation. However, if the Symbiodinium is dissociated from the coral cells for a longer period of time, the build-up of toxins will cause cellular death and degradation of the Symbiodinium. When the Symbiodinium is associated with the coral, MAMP/PRR interactions will activate an inducible promoter to produce an anti-toxin. This anti-toxin neutralises the toxin to allow the Symbiodinium to continue normal nutrient exchange while in association with the coral cell.
A potential toxin/antitoxin system would be the CcdA/CcdB pair in which the CcdA toxin is neutralised by the CcdB antitoxin (Afif et al., 2001). CcdA inhibits the bacterial DNA gyrase in E. coli. DNA gyrase is a type II DNA topoisomerase enzyme which is mostly found in bacterial species. However, one study has found ATP-dependent topoisomerase activity in C. reinhardtii, a eukaryotic cell, indicating potential structural and functional similarity with DNA gyrase (Thompson and Mosig, 1985). As such, it is possible that CcdA will be able to inhibit this topoisomerase in C. reinhardtii and thus decrease translation of its DNA. Since the activity of this particular enzyme targets chloroplast, the toxin/antitoxin system would be a practical biocontainment solution since the toxin can inhibit the microorganism’s photosystem, and starve the cell by disabling its photosynthetic machinery (Afif et al., 2001). Characterisation of this ATP-dependent topoisomerase will be a necessary step in this proposed skill-switch.
While literature surrounding coral/symbiont recognition and phagocytosis mechanisms is limited, some recent studies have identified mannose recognising Lectin ConA as a conserved MAMP across Symbiodinium sp. (Logan et al., 2010, Wood‐Charlson et al., 2006). Lectin ConA has been implicated in key signalling pathways within other eukaryotes (Fu et al., 2011, Sina et al., 2010) and this makes it an ideal candidate for a potential kill switch within our modified Symbiodinium.
Due to the genetically-modified nature of our proposed system, it is important to understand the safety concerns behind it and to address them. Introducing the genetically modified algae Symbiodium goreaui in the Great Barrier Reef without further experimentation would be unwise and may potentially have negative consequences. For example, introducing genes that appear favorable in the environment can result in other genes that were considered unfavorable to be lost from the gene pool, reducing genetic variability. However, in the face of environmental change, this can prove detrimental to the survivability of the algae and hence to the survivability of the coral, leading to greater damage than was initially anticipated. For this reason, Phase III of the project will focus on mechanisms for the safe introduction of the algae in the environment.
Our current proposed implementation approach aims to enhance the coral-algae interaction. This will ensure that once the algae are introduced in the environment, it will bind to the target coral host and maintain the symbiotic relationship. Of interest is the lectin-glycan interaction explored below.
There are complex interactions between algae and the coral host that ensure the maintenance of the symbiotic relationship. A proposed model involves interaction between the corals and the symbiont through microbial-associated molecular patterns (MAMPs) and pattern recognition receptors (PRRs; Tortorelli et al., 2021) to provide a beneficial symbiosis. One such model involves lectins. Lectins are able to recognise and bind carbohydrates on the surface of cells (Sharon and Lis, 2004). Algae may express glycan polymers on their surface that bind to lectins on the surface of corals in a lock-and-key mechanism (Tortorelli et al., 2021). Tortorelli et al. (2021) utilised the Anemone coral models E. diaphana of the Great Barrier Reef and 3 Symbiodiniaceae algae, including C. goraui, B. minutum and F. kawagutii for their study. The cell wall monosaccharide profiles of the algae were determined which showed that the cell-surface glycan profile differed between the three algal species. Notably, the cell walls of the 3 algal species contained glucose and D-mannose. It was found that lectin ConA is able to bind specifically with the D-mannose on the surface of all three algal species (Tortorelli et al., 2021). The authors postulated that D-mannose and D-glucose on the surface of symbionts may be important for their recognition by the coral and in retaining the symbiotic relationship once bound. Investigating whether Symbiodinium goreaui possess D-mannose and D-glucose on their surface will be important to ensure that the symbiotic relationship of the introduced algae and the coral is maintained.
Similarly, lectins have been discovered in many algal species (Hwang et al., 2020). A mannose-binding lectin isolated from the algal species G. chiangii has binding affinity for the following monosaccharides: β-Glc-sp, β-Gal-sp, α-Man-sp and Maltotetraose-β-Sp (Hwang et al., 2020). It is important to mention that the lectin from G. chiangii was stable at a wide range of temperatures even at 90 °C at which temperature it retained about 15% of activity (Hwang et al., 2020). Future research will determine whether corals possess β-Glc-sp, β-Gal-sp, α-Man-sp or Maltotetraose-β-Sp on their surface which will enhance binding to the algal lectins, improve the interaction and prevent the expulsion of the algae. Considering the increasing temperatures of oceans, the lectin from G. chiangii could have great potential due to its relative stability at higher temperatures.
Overall, the lock-and-key mechanism proposed by Tortorelli et al. (2021) and the cross-talk between lectins and glycan polymers will ensure that the coral interacts only with specific glycans on our introduced algae and not other symbionts. The lectin-glycan interaction should be further explored in the future as part of our proposed implementation to ensure the introduced symbiont is not expelled from the corals.
The greatest challenge faced during our iGEM project was the continuation of lockdowns in Australia which significantly limited our ability to conduct lab work. The COVID-19 lockdown occurred in June and continued to October with our university labs closing in line with health policy. We had planned our lab timeline with a start date of early June and projected to spend 4 months conducting lab work. However, our timeline completely changed because of continuing lockdowns with no certain end date in sight. This also made it difficult to secure lab equipment and we were unable to access algal species for our experiments.
To overcome this limitation, we needed to devise a new plan forward in an uncertain environment. We shifted the focus of our project by devoting more time and resources towards:
- Dry Lab: Conducted modelling to understand how our genetically engineered algae could be heat resistant in the GBR.
- Human Practices: Innovative effort to engage the wider public about our project through creating a survey and consulting a greater pool of stakeholders.
- Science Communications: Created a highly interactive array of education materials, in the form of a board game and Indigenous virtual art exhibition.
Through our discussions with social scientists and the wider public, we realised there was considerable debate surrounding the ethics of introducing genetically modified species into nature. At the current pace of technological advancements, there has been limited time for ethicists to properly weigh up the morality of synthetic biology solutions.
The development of new “living machines” has opened up discussion from ethicists about whether humans can be creators of novel forms of life (Boldt, 2019). Redesigning an organism can fundamentally alter an ecosystem and our approach to life. Boldt discussed how there would be a marked paradigm shift from synthetic biologists being considered as researchers, to now being regarded as “creators” (2019). The ethics of humanity moving from a small act of manipulation to an entire reinvention of nature, is a point of debate for social scientists and a concern raised by wider society in our survey.
There are ethical concerns surrounding the potential safety risks of genetically engineered species. Dr Boldt highlights that while democratizing synthetic biology may result in novel, innovative applications, there are still issues raised over the deliberate misuse of this technology (2019). Potential scenarios may involve uncontrolled gene replication or new organisms being leaked into the outside environment from research labs (Boldt, 2019).
Ultimately, we learned these biosafety concerns and ethical values should be better communicated to the public and not oversimplified by researchers. The public is interested in learning more about ethical frameworks and as such, it is our duty to ensure the public is well informed about our risk mitigation strategies and consideration of ethical values.
Social Concerns & Public Acceptance
Our survey was centered on understanding public perspectives towards our synthetic biology solution and it was critical to learn more about social concerns without any preconceived biases. In order for us to successfully implement our solution, it must reflect the values and needs of wider society. In the survey, we discovered the majority of participants wanted to provide detailed feedback about the perceived safety concerns of our solution. We discovered the most common areas we should focus on were:
- Adverse impacts of genetic engineering on the marine environment and ecosystem dynamics
- Long term or unforeseen risks of genetic engineering
Our Risk Management framework addresses feedback surrounding environmental impacts and any long term risks of genetic engineering. Under the section Safety and Concerns, we have proposed the key perceived risks of our solution which are in line with the insights gathered from the public and stakeholders, along with ways we have attempted to mitigate these risks.
In the future, we hope to grow the public’s awareness through interactive, educational mediums and continue to facilitate a two-way dialogue that can shape our project and address their aforementioned concerns. This would help address the disconnect between the knowledge of the public and the science, in order to allow people to make informed decisions about the implementation of genetic engineering.
Through our discussion with Traceylee Manuwuri Forester, we recognised the importance of researchers fostering open communication with Indigenous communities at the earliest stages of the research trajectory. Traditional Owners have a deep spiritual connection to the land that we need to respect and be mindful of as we design our solution. Essentially, we cannot implement our synthetic biology solution without the permission of Indigenous communities. It is imperative we engage early and seek to blend the voices of Indigenous peoples into our solution to ensure it is socially responsible and good for the world.
Our Next Steps
Scientific research is known to be an iterative process - changes are constant, and our team is aware that it is difficult to plan out the future without expecting any bumps in the road! However, we felt an immense sense of responsibility to provide the public with a comprehensive pipeline that delves into the future endeavours of PROTECC Coral.
Having had two years to develop the theory behind our project, we hope that the next year allows for us to put theory into practice and continue with the research and development stage. Although we have the brightest minds working on this project at UNSW, the time taken to create genetically modified organisms are known to be notoriously long, due to the thorough testing it has to undergo before it is eligible to go through the application process at the Office of Gene Technology Regulator (OGTR). We hope to engage with the OGTR in the second half of the research and development phase, as their guidance could help us navigate the intricacies of our project and provide the appropriate services to tackle any hurdles we face along the way.
After being approved for the aforementioned licence, we would like to partner with other government-affiliated organisations such as the Australian Institute of Marine Sciences (AIMS), to explore the possibility of using SeaSim - Australia’s leading facility that provides its researchers with the opportunity to scale their laboratory research and unlock valuable insights into how their creation interacts with other organisms in a simulated environment (Australian Institute of Marine Science, 2016). Kevin Gale mentioned that the introduction of a genetically modified organism into our natural environment is akin to letting a genie leave the bottle - we cannot place it back in its lamp after it has been released. Thus, we expect that this stage of ex-situ testing to be an arduous, time-consuming process. Ultimately, this is the process that needs to be followed to develop a solution that is good and responsible for the world.
If our genetically modified product is shown to thrive in this environment and has positive indications of being transferred to an in-situ environment, we would apply for the research permit at the Great Barrier Reef Marine Park, which would allow us to conduct on-sight testing. It would be in our best interest to apply for a zone that has already lost a portion of its biodiversity, as this would be the safest way to implement our engineered Symbiodinium into the ocean.
As mentioned earlier, we expect deviations from this plan due to the certainty of change, but we hope that our efforts at creating a transparent and detailed blueprint does not go unnoticed by the masses. As the years progress, we will strive to revisit this plan and continuously update our most important stakeholders, the general public, regarding our solution.
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