Team:UNSW Australia/Implementation

Proposed Implementation: PROTECC Coral

Implementation takes scientific research and applies it into the world for the benefit of our communities and ecosystems. The devastating impacts of climate change, particularly on the Great Barrier Reef and reefs worldwide have seen a shift in public attitudes towards greater acceptance of synthetic biology solutions. Nevertheless, there are concerns that must be addressed when proposing to implement a genetically modified solution into a natural environment. It is important that our team considers the technical, safety, ethical and socio-cultural concerns.

By looking at the existing implementation methods others have applied in conservation efforts around the world, we can establish the ‘safest’ delivery of our project into the real world. This can help us ensure our project is responsible and good for the world. Through consultations with stakeholders and experts who have experience in coral conservation, we were able to ideate a realistic implementation of our solution.

The proposal to implement a synthetic biology solution will often occur over an extended period of time, in order to ensure the above considerations have been met. The UNSW iGEM team’s solution involves two key phases to take place over two years, culminating in implementation of the designed solution in ex-situ tanks. The University of New South Wales is fortunate enough to have access to research tanks at the Sydney Institute of Marine Science (SIMS). Looking forward, the next step would involve the transfer of these genetically modified corals to an in-situ marine environment.

Our Proposed End-Users & How We Envision Others Using Our Project

The UNSW iGEM team defines ‘proposed end user’ as the party which will implement the solution, bringing it into the real world. Our proposed end users are governments and NGOs, who would deploy our synthetic biology solution as a means to protect the coral reefs from rising ocean temperatures.

Professor Matthew Kearnes, a social scientist and ethicist, urged us to deeply consider who our proposed end user should be. What are their incentives? To whom are they accountable? Would the general public and stakeholders accept them as the deployers of a synthetic biology solution in our natural environments?

We propose that the end user’s incentive should not be profit, but a desire to protect the coral reefs from climate change. Nature, and by extension the coral reefs, belong to everyone. As such the proposed end user should be accountable to the people. Next, in order for stakeholders to accept anyone holding this responsibility, they must have confidence that their voices will be heard and integrated into the implementation of our solution. Finally, the proposed end user must have the scientific capacity, resources and authority to responsibly deploy this solution for the good of the coral reefs.

Our identified profile for the ‘ideal’ end user leads us first to the government. The government is accountable to the people, and has a duty to listen to and act in the interests of the community. Furthermore, The Great Barrier Reef Marine Park Authority (‘GBRMPA’), a specialised government agency, has an interest secured by legislation to act in the best interests of the reef. (1) The GBRMPA is a well respected agency, has vast experience in coral reef conservation and has a history of incorporating stakeholder voices. (2)

Our second proposed end users are non-government organisations (NGOs), who play a major role in the stewardship of natural environments such as coral reefs around the world. These organisations are non-profit, which align with our team’s intention to use our synthetic biology solutions in an open source manner. Further, international NGOs such as The Nature Conservancy, WWF and UNESCO are able to effectuate real solutions beyond Australian borders. Their global reach would allow this solution to benefit coral reefs and communities around the world. Finally, the independence of NGOs from government, may also be conducive to a more straightforward path to action, as they are less likely subject to the sway of political change.

After our consultation with Dr. Aditi Mankad, a senior CSIRO research scientist, we gained an appreciation for how synthetic biology can be used as a part of a greater whole. We thus envisioned our synthetic biology solution to work alongside traditional coral conservation methods, such as coral replanting. Moreover, we wanted our solution to be open-source, so that we could share our methods and results with the wider scientific community. The UNSW iGEM team truly believes that through a global multifaceted and multidisciplinary effort, humanity can protect the coral reefs. Our greatest hope and vision for the future, is a world where the coral reefs may survive and thrive forevermore.

How We Would Implement Our Project In The Real World

Ex-Situ Uptake & In-Situ Implementation

Following our consultations with Melissa Katon, our team gained greater insight into the potential of ex-situ farming of our modified Symbiodinium model. Melissa discussed with us the use of research tanks at the Sydney Institute of Marine Science (SIMS) to allow for ex-situ coral rehabilitation. The University of New South Wales is fortunate enough to have access to these research tanks. These tanks imitate the in-situ environment as sea water from the ocean is directly circulated within.

Looking forward, the next step would involve the transfer of these genetically modified corals into an in situ marine environment. Speaking with Lawrence Menz greatly steered our focus towards the considerations that must be had prior to implementation of a project in an in situ environment. He encouraged us to implement our solutions in locations that have zero, or close to zero, reef, in order to mitigate potential negative impacts on biodiversity.

Our team wanted to ensure that our proposed implementation was responsible and good for the world. With great consideration into the values and needs of stakeholders and the greater community, our team designed a proposed implementation that was intended to be safe and sensitive to risk. Our three-stage approach was designed to mitigate risk to the corals, the surrounding biodiversity and the ecosystem as a whole.

  1. Ex-Situ Testing
    • Long-term observation of modified Symbiodinium impacts on the coral and greater biodiversity, within a controlled and contained environment simulating ocean conditions.
  2. 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.
  3. 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.

Licensing & Permit Applications

To test our engineered Symbiodinium in-situ, several applications would have to be undertaken in order to gain permits for on-site testing:

  1. The Great Barrier Reef Marine Park Authority requires research applicants to justify the reasons as to why certain locations of the reef were chosen, as well as our proposed route of research. As explained by Lawrence Menz, the best and safest way to implement our engineered Symbiodinium into the ocean is to release them to a part of the reef that has already lost its biodiversity. (3)
  2. It is also important to comply with the Office of the Gene Technology Regulator (‘OGTR’) standards of risk analysis and regulation of our solution: “Genetically modified (GM)… crops grown in Australia were approved for commercial release only when the regulator found that the GM crops were safe for human health and the environment as non-GM versions”. (4)

Social Licensing

The successful implementation of our synthetic biology solution also rests heavily on the concept of “social licensing” that we discussed with Associate Professor David Suggett. The social license may be equally important, or perhaps even more so, than any other type of formal license or permit. Through our consultations with social scientists, we learnt about the disconnect that is often present between scientists and people, as scientists may define the problem without engaging in stakeholder consultations. In order to gain a social license, it is necessary that outreach to and involvement of stakeholders are meaningful and considered at every part of the research and implementation trajectory. Conversations with our stakeholders: Traditional Owners, Biodiversity Experts, Biospectors, Coastal Protection, and Tourism & Commercial and Recreational Fishing, would be sustained as part of our proposed implementation. These conversations would allow us to respectfully engage with a diverse range of needs and values.

Our proposed implementation would involve a continuation of the human-centred design process that we adopted in our IHP journey. Another facet of social licensing involves the complex dynamic between the public and synthetic biology, amidst the evolving Australian climate change conversation. In our conversations with Dr. Aditi Mankad, she spoke about the national value that is found on the Great Barrier Reef, which is contributory to a surprisingly risk-positive general public. However, there remains a degree of public distrust towards novel technologies, particularly on the fragile coral reef that remains on the Great Barrier Reef. Therefore, it is important that our proposed implementation incorporates science communication, to educate the public about the dying coral reefs, and the synthetic biology solutions that have much potential to save it.

The Safety Aspects We Need To Consider: The Kill Switch

With any proposed release of GMOs, a major public and scientific concern is the unintended invasion of the species outside of its intended environment. This may cause changes in ecosystem dynamics and inadvertently result in decreased biodiversity rather than conferring the intended function. In order to limit the risks of this occurring, biosafety considerations can be integrated into project design and can inform experimental approaches. Genetically modified microorganisms can be designed with biocontainment systems such as kill switches or natural/synthetic auxotrophy. These will result in cell death upon exposure to certain environmental conditions, whether that be the presence of a specific molecule or a change in pH, and thus prevent integration into other species.

As existing biocontainment systems for eukaryotic algal symbionts are limited, our team conducted research to design a novel biocontainment system specifically for Symbiodinium. It aims to take advantage of natural mechanisms by which Symbiodinium associates with 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. (5) 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. (6)

Our kill switch will use this mechanism to activate a toxin/anti-toxin system. The toxin will be constitutively produced under a weak promoter, allowing for Symbiodinium to survive for some time even when detached from a coral host. This allows for a short window to introduce modified Symbiodinium to corals during either ex-situ or in-situ implantation. However, if the Symbiodinium is dissociated with the coral cells for a longer period of time, the toxin build up will cause cellular death and degradation. When associated with the coral, MAMP/PRR interaction will activate an inducible promoter to produce an anti-toxin. This will neutralise the toxin and allow the Symbiodinium to continue normal nutrient exchange 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. (7) 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. (8) 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. (7) 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. (9, 10) Lectin ConA has been implicated in key signalling pathways within other eukaryotes. (11, 12) This makes it an ideal candidate for a potential kill switch within our modified Symbiodinium. However, due to limited current knowledge on Symbiodinium cellular mechanisms, Phase II of our project will focus on identifying a suitable Lectin ConA associated signalling pathway to be modified in order to implement our kill switch. This will therefore build upon our Phase I design to undertake the build and test experimental aspects, allowing for validation of this biocontainment strategy.

Figure 1: Diagram representing a potential toxin/antitoxin kill switch to be implemented within Symbiodinium. Figure created in BioRender.

Other Challenges To Be Considered

COVID-19 Limitations

The most prominent challenge faced during a global pandemic is the limitation brought upon the scope of our lab work. Therefore, the continuation of lab work in Phase II of our project will be more extensive, assuming COVID-19 limitations are lifted. Despite these challenges, the relationships and insights we have established with stakeholders and coral experts during Phase I of our project will be useful for the workflow of the incoming iGEM 2021 team, and guide their approach to lab work.

Selection Of The Correct Coral Species

From the advice gathered in Phase I of our project, our future team will have to consider what species of coral they would like to work with. From the advice gathered this year, it is best to work species of coral which are unprotected, or common coral species which are used in most coral research work. General lab details, such as updates to the safety form, must also be considered when working with coral species, as lab work will be more complex and new ethical concerns will have to be considered.

The Greater Biodiversity

In our stakeholder consultations, we spoke with Lawrence Menz, a marine biologist who is a member of the Maldives Underwater Initiative (MUI), a marine conservation initiative. In our discussions, Menz spoke about how biodiversity concerns are at the forefront of any coral restoration project. The greater biodiversity of the coral reef and ecosystem, including diverse marine species, the more resilient it is to stresses such as coral bleaching events. Menz spoke about the importance of ensuring that all present corals are able to uptake the genetically-modified microalgae. Such considerations would prevent one species of coral obtaining an advantage over another, thus resulting in loss of biodiversity over time. Menz suggested it was optimal to implement synthetic biology solutions in locations that already have zero, or close to zero, reef, in order to mitigate potential negative impacts on surrounding biodiversity.

Ethical Concerns & Animal Welfare

Revive & Restore is an organisation dedicated to enhancing biodiversity by incorporating biotechnologies into standard conservation practice. In our conversations, Revive & Restore spoke about the importance of animal and environment welfare, given the rich biodiversity on the Great Barrier Reef marine ecosystem. We learnt that welfare issues may arise in the ex situ and in situ transfer of corals, microalgae and associated living matter. Ben Novak, from Revive & Restore, was kind enough to send us his paper and discuss with us the concept of animal distress as inherent in terminal intervention, and indeed as part of ecology. Thus, in the same way distress drives evolution and adaptation, brief moments of distress caused by our proposed implementation, may be justified to confer future, compounded, benefits on the ecosystem. (13)

Social Concerns

In subsequent conversations with Lawrence Menz, warned how the creation of a synthetic biology solution, and its implementation into the natural environment, may cause the public to place less importance on preventing the coral bleaching problem in the first place. A common concern in conservation projects involves the argument that people may continue to engage in activities that contribute to the problem, if the consequences of their actions are “solved”. This is a major challenge as the overarching cause of coral bleaching- climate change- remains a major problem. Climate change requires an international social and political effort, which is yet to be seen. A challenge in our proposed implementation would be to frame the problem, such that efforts to combat climate change are still valued by the public.

Social Licensing & Public Acceptance

In our discussions with Associate Professor David Suggett, he introduced us to the concept of “social licensing”, which impacted the way in which our team perceived the relationship between the public and science. A major component of the success of a proposed implementation, lies in the level of acceptance the public feels towards the technology being implemented. It lies in questions about what is being implemented, in what manner, and what are the extenuating effects. Therefore, overcoming this challenge would mean greater involvement and education of the public and relevant stakeholders at the earliest stages of the research trajectory. Science communication would be best utilised in demystifying the traditional stigma surrounding intervening technologies. Possessing an understanding of the social landscape, and the needs and values of people, are crucial in proposed implementation considerations.

Our Contribution Towards Global Conservation Efforts

An important consideration in our proposed implementation is being able to place and frame our synthetic biology solution within a larger context of other coral conservation efforts. Climate change is a real and pressing global issue that has already caused much devastation to environments worldwide. Therefore, we see our solution, and its proposed implementation, as our contribution towards a larger global effort to conserve species and the natural environment. In our discussions with Dr. Aditi Mankad, a social research scientist, we explored the potential of synthetic biology as a “novel tool in our toolbox”. We discussed the potential of synthetic biology when used alongside other coral conservation efforts, such as coral nurseries and replanting. We believe that our solution, and its proposed implementation, can enrich the efforts to protect coral worldwide.

References

  1. Great Barrier Reef Marine Park Act 1975 (Qld) s 2A
  2. Great Barrier Reef Marine Park Authority. Indigenous Reef Advisory Committee [Internet]. [place unknown]: Australian Government; 2020 [cited 2020 Oct 24]. Available from: http://www.gbrmpa.gov.au/about-us/reef-advisory-committees/indigenous-reef-advisory-committees
  3. Great Barrier Reef Marine Park Authority. Checklist of Application Information. [internet]. [place unknown]: Australian Government; [date unknown] [updated 2020 Sep 3; cited 2020 Oct 25]. Available from: http://elibrary.gbrmpa.gov.au/jspui/bitstream/11017/3647/1/Checklist-of-application-information-research.pdf
  4. Office of Gene Technology Regulator. Genetically modified (GM) crops in Australia. [internet]. Canberra, ACT: Australian Government; [date unknown] [cited 2020 Oct 25]. Available from: http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/content/9AA09BB4515EBAA2CA257D6B00155C53/$File/11%20-%20Genetically%20modified%20(GM)%20crops%20in%20Australia.pdf
  5. Liu H, Stephens TG, González-Pech RA, Beltran VH, Lapeyre B, Bongaerts P, Cooke I, Aranda M, Bourne DG, Forêt S, Miller DJ. Symbiodinium genomes reveal adaptive evolution of functions related to coral-dinoflagellate symbiosis. Communications biology. 2018 Jul 17;1(1):1-1.
  6. Davy SK, Allemand D, Weis VM. Cell biology of cnidarian-dinoflagellate symbiosis. Microbiol Mol Biol Rev. 2012;76(2):229-61.
  7. Afif H, Allali N, Couturier M, Van Melderen L. The ratio between CcdA and CcdB modulates the transcriptional repression of the ccd poison–antidote system. Molecular microbiology. 2001 Jul;41(1):73-82.
  8. Thompson RJ, Mosig G. An ATP-dependent supercoiling topoisomerase of Chlamydomonas reinhardtii affects accumulation of specific chloroplast transcripts. Nucleic acids research. 1985 Feb 11;13(3):873-91.
  9. Logan DDK, LaFlamme AC, Weis VM, Davy SK. 2010. Flow cytometric characterizaion of the cell-surface glycans of symbiotic dinoflagellates (Symbiodinium spp.). J. Phycol. 46:525–533
  10. Wood-Charlson EM, Hollingsworth LL, Krupp DA, Weis VM. Lectin/glycan interactions play a role in recognition in a coral/dinoflagellate symbiosis. Cell Microbiol. 2006;8(12):1985-93.
  11. Fu LL, Zhou CC, Yao S, Yu JY, Liu B, Bao JK. Plant lectins: targeting programmed cell death pathways as antitumor agents. Int J Biochem Cell Biol. 2011;43(10):1442-9.
  12. Sina A, Proulx-Bonneau S, Roy A, Poliquin L, Cao J, Annabi B. The lectin concanavalin-A signals MT1-MMP catalytic independent induction of COX-2 through an IKKgamma/NF-kappaB-dependent pathway. J Cell Commun Signal. 2010;4(1):31-8.
  13. Fischer B. The Routledge Handbook of Animal Ethics [Internet]. New York: Routledge; 2019. Chapter 21, Building Ethical De-exctinction Programs Considerations of Animal Welfare in Genetic Rescue. [cited 2020 Oct 6]. Available from: https://www.researchgate.net/publication/339292855_BUILDING_ETHICAL_DE-EXTINCTION_PROGRAMS_Considerations_of_Animal_Welfare_in_Genetic_Rescue