Future Directions for CORA ANU

As the project continues to develop and advance, we want to maintain the connections we have developed thus far and ensure that local communities and historically marginalized First Nations’ voices are centered in all phases, from scientific development, to policy, and end-goal implementation. We hope that our survey investigating public perceptions to synthetic biology for coral bleaching remediation is approved by the human ethics research committee and upon approval, that we can engage with local community groups on the reef to disseminate the survey. The results of this will provide key understandings about how the public engages with synthetic biology and what role they play in policy surrounding conservation, and build upon prior work. By understanding these issues, we hope that CORA is a human-centric project, encapsulating salient issues for the public. Our framework POLYP will be a valuable tool in guiding these discussions and will ensure that research is guided by key community values in its impact, end-users, and future implementation.

Impact

The Great Barrier Reef is the world’s largest coral reef ecosystem, stretching more than 2300 km along the coast of Australia [1]. This diversity includes but certainly is not limited 410 species of hard coral, 1,620 species of fish, 2,000 species of sponge, and 500 species of marine alga living across 70 bioregions, making it the most biodiverse World Heritage Site [2, 3, 4]. Additionally, it forms a key part of the Australian global identity, and has deep cultural ties to the Traditional Landowners of the Sea Country. Thus, we recognize that the impact of CORA is not limited to the organisms who inhabit the reef, but also the people whose livelihood depends on the reef.

Our research hopes to go beyond a scientific contribution towards amelioration of coral bleaching, but also to provide insights into synthetic biology more broadly. Given the nascent nature of synthetic biology as a field, researchers are somewhat limited in the scope of application (e.g. limitations of engineering certain organisms. As future phases of the project look away from proof of concept in E. coli and towards a different chassis, that may include bacteria native to the coral surface mucus layer, we aim to expand the genetic engineering toolkit available to synthetic biologists, thereby broadening the scope of synthetic biology solutions.

We also hope that our project will have an impact on coral science more broadly, by delving into symbiotes and mechanisms of coral bleaching. Finally, by designing our own approach for public engagement and reflection, are providing a framework for researchers and even policy makers to utilize when considering synthetic biology solutions, and how these may interact with public perceptions and appropriately acknowledge the significance of the GBR to our stakeholders. Of course, we recognize that CORA is not the sole solution to the salient issue of coral bleaching, but we hope that by furthering the available options to conservation, we have expanded the toolkit the government can employ in taking action against environmental harms from climate change.

End Users

Another important part of the product development of a synthetic biology solution is giving consideration to who the ‘end user’ of your solution would be. That is, who would actually use the product when the engineering is all said and done. We propose that the end-users for CORA are government agencies and non-government originations (NGOs). We believe that these end-users meet some desirable criteria. First, they are accountable to people connected to the GBR. Notably, we are ruling out end-users who are motivated by profit, and choosing not to commercialize our project. We want our end-user to share the same values as us, most importantly a desire to protect the coral reefs from climate change. Second, we wanted our end-users to have the capacity to ensure that stakeholder’s voices are promoted and integrated into the implementation of CORA.

The Great Barrier Reef Marine Park Authority (GBRMPA) is a specialized government agency with the legislated mission goal of acting in the best interests of the GBR. They work with a range of partners, advisors, and stakeholders to manage and conserve the GBR. Further, they permit scientific research groups to conduct often small-scale experiments that further this goal. Given their status as a government agency they are accountable to the people and ought to work in the best interests of the community. As such, we identified them as a good end-user for CORA.

Beyond government, synthetic biologists in conservation can look to NGOs as a means of implementation. NGOs are inherently operated in a not-for-profit way, and therefore align with our intention to not commercialize CORA. Further, NGOs can operate internationally, thereby expanding the scope of CORA’s implementation to reefs beyond that of the GBR who are also undergoing similar mass bleaching events. Another important aspect of NGOs is that they are largely independent from the government, and thus provide a straightforward path of action that is less influenced by political agendas or legislative bureaucracy.

Our discussion with Dr. Line Bay prompted us to think about the end-users of CORA beyond the two we had already identified. She pointed out that a full-package implementation of any synthetic biology solution should position the local community. This led us to think about citizen science as a means of implementation. Broadly, citizen science refers to research conducted with participation of the general public. Such platforms already exist in Australia, with CSIRO coordinating different efforts to issues of marine debris and bushfires [5]

This is also important in increasing the accessibility and inclusivity of our science. As such, we envisage that future iterations of our design cycle would take further steps to address concerns of biosafety (i.e. through implementation of a kill switch)

Implementation

Central to synthetic biology is real-world implementation of engineered solutions. However, deployment of engineered microbes in large scale and in open environments is not without risk; gene transfer may confer antibiotic resistance

Therefore, in almost all nations the introduction of genetically modified organisms GMOs into the environment must comply with strict regulations. The ultimate aim of our project is for real-world deployment of our engineered bacteria into coral reefs, in order to target factors influencing coral bleaching in situ. However, this application raises concerns surrounding biosecurity and biocontainment.

By looking at existing implementation methods of conservation solutions and some of the regulations facing Australian researchers, we can devise a ‘safe’ implementation pipeline for CORA in order for large-scale application to be feasible. This will help to ensure that our solution is responsible and good for the world.

1. Ex situ testing

This initial step would involve long-term observation of CORA in a controlled environment and contained environment. This would involve work with collaborators, for instance at the Australian Institute of Marine Science’s National Sea Simulator.

2. In situ testing in areas of low biodiversity

This would involve long-term observation of CORA’s effects on coral and relative ROS levels in a area of the reef with low biodiversity. This would be small-scale and would need to be removable, and thus not posing a significant risk to the surrounding biodiversity.

3. In situ release of CORA

This would involve the release of our modified bacteria into the natural environment with normal biodiversity. This would require continual observation and monitoring of the environment.

In order to test CORA in situ, an important part of our implementation strategy, we would have to deal with several regulatory bodies.

1. The Great Barrier Reef Marine Park Authority (GBRMPA) requires research applications that would involve environmental impact statements and public environment reports for longer-term studies [6]. In our conservation with her, Dr. Line Bay noted that this step is a current roadblock for research, and the best way forward is to work with the GBRMPA to co-design solutions.
2. We would further need to comply with the Office of the Gene Technology Regulator (OGTR) standards of risk analysis and approval of any GMO would require an intensive process of public consultation. Further, GMO stewardship standards the OGTR advocate require responsible management of biotechnology from their discovery and development to their use and eventual discontinuation.

Discussions about implementation helped us refine our design strategy for future phases of the project. The concept of stewardship in particular prompted us to reconsider our design by prompting to address concerns of biosafety and responsible management immediately, by further engineering our bacterium to introduce a kill switch.

Kill Switch

Given that our proposed implementation involves the release of a genetically modified organism into the environment, a major biocontainment concern is the unintended localisation of our modified species outside its intended environment. Without successful biocontainment techniques there may be changes to the marine ecosystem dynamics and the loss of biodiversity. One approach to biocontainment is usage of a kill switch. A kill switch is a gene circuit that is used to maintain essential gene expression or block toxin gene expression when under the biocontainment conditions [7]. Upon loss of this biocontainment signal, the circuit either blocks the production of essential components or induces toxin gene expression in order to kill the cell, thereby enabling controllable deployment of synthetic biology solutions.

Because of time constraints and challenges encountered in the lab, we were not able to engineer a kill switch into our bacteria. As the kill switch is somewhat independent to the main aim of our project in its current phase, and much more of a future implementation consideration, we instead chose to devise a proposed gene circuit. In this project we used prior teams work to inform our design. In particular, the kill switch database by 2016 Marburg and the reference table provided by 2020 Fudan gave us a good starting point to look at existing kill switch approaches [8,9].

The kill switch needs to be highly lethal in non-permissive conditions but stable avoid in permissive conditions to not undermine survival and implementation. One method for a kill switch is using a molecular ‘sponge’ model based on toxin/antitoxin pairings to sequester the toxin and prevent cell death. Regulation is achieved through the use of environmentally sensitive promoters and constitutive promoters. In future design iterations, we plan for CORA to contain a kill switch based on the ccd gene system with the toxin/antitoxin pair of ccdB/ccdA [10]. The toxin protein ccdB that acts as an inhibitor for the GyrA subunit of DNA gyrase, causing arresting it after the double-stranded break and thereby causing cell death. ccdA encodes for the antitoxin CcdA, which binds to CcdB and prevents it from inhibiting DNA gyrase, thereby facilitating cell growth. Thus, whether a bacterium lives or enters a quiescent state depends on the relative concentrations of CcdA and CcdB, and therefore the use of an environmentally sensitive promoter to upregulate the excess production of the toxin in impermissible conditions. This principle is shown below:

We initially identified using a cold-acting promoter cspA so that the kill switch becomes activated when temperature decreases (with full activation occurring at 15C). However, during our chat with Bioplatforms Australia, we were notified of their work in sequencing data of coral host species and symbiodinium [11]. We hope that future collaboration could help to identify and utilise in our gene circuits better promoters that are more responsive to the reef environment.

Of course, our work with E. coli for CORA has been proof-of-concept, and so it is important to note that moving kill switch parts that were developed and tested in E. coli into alternative cellular hosts is a difficult process, and can undermine kill-switch performance. This is an important future consideration; while our solution was developed and tested in E. coli, it may be the case that implementation is best in another organism. Furthermore, synthetic biology solutions developed for environmental applications will ultimately be deployed into contexts with unpredictable factors and stressors that may affect the gene circuit function.

Integrated Human Practices

A synthetic biology solution is one of a plethora of solutions being utilised in the fight against coral bleaching. However, it is essential that any solution is responsible and ultimately good for the world. Following on from our conservations with academics, peers, and social scientists, we wanted to ensure that feedback from stakeholders was successfully incorporated into our design process. In particular, our conservation with Dr. Line Bay elucidated some key considerations about science communication and policy, and we sought to include these takeaways into our own approach.

So, what does a ‘good’ approach to human practices look like? Initially daunted by human practice, this was a key question for our team. Given this, we also decided to take a somewhat unique approach to integrated human practices; in line with the iGEM ethos of building upon past work and making contribution to future teams, by integrating an analysis of past iGEM teams’ approaches to human practices, we wanted to devise our own framework help inform future teams about how to approach human practices. Not only did we think about whether our scientific work, CORA, was good and responsible, but we were equally concerned whether our devised human practices framework POLYP was ‘good’ for society. We hope that POLYP becomes a useful tool and is carried forward into new synthetic biology projects!

Meta analysis

As discussed above, human practices was an aspect of the project that we were unsure how to best approach. We recognized that human subjects research is based upon activities such as informal conversations, public engagement, structure consultations, and large data surveying. However, as our team predominantly has a background in science and not human subject research, we were daunted by how best to approach this part of iGEM.

In line with the iGEM ethos of collaboration, we decided the best place to start would be the efforts of past teams. As we started to look at the contributions and approaches of these teams, we began to notice the diversity in approaches employed. This led us to a few key questions. What can we learn from different methods used to address Human Practices? How do these methods differ across different applications of synthetic biology? How does a team best identify relevant stakeholders and ensure that their perspectives are appropriately engaged with? Ultimately, we wanted to answer the question of what a ‘good’ approach to Human Practices looks like.

In order to address these questions, we decided to perform a mini meta-analysis. We started by identifying previous award winners and teams who had unique or distinct approaches to human practices. As shown in the figure below, we wanted to include a diverse mix of teams from different geographic regions (to account for differences in cultural norms) and from a range of iGEM villages (to account for differences in end-users and implementation). While it is important to note that this analysis is by no means exhaustive, it helps to paint a clearer picture of what a universal ‘good’ approach might be.

Approaches to HP

From our meta-analysis, we found that human-centered design was a core tenet shared between iGEM teams. However, a point of difference between teams was the use of a specified framework to guide their human practices. Several of the teams we looked at (e.g. Exeter 2019, TU Eindhoven 2022) employed the AREA framework, a cyclical method for incorporating relevant community and expert opinion into a project [1, 2]. This framework was developed by UK Research and Innovation and has been endorsed by the European Commission as a means for researchers to demonstrate awareness of and commitment to the principles of responsible research and innovation [3]

Anticipate involves describing and analyzing the intended and unintended impacts of our project. Reflect encourages consideration of the motivations of the research in light of the potential implications and associated uncertainties. Engage involves opening up these discussions to broader deliberation through consultation of expert and public opinion. Act involves using these processes to direct the trajectory of the research and innovation process itself. UCopenhagen 2020 took the principles of this framework to make their own framework, titled CID, standing for Considerations, Interview and Decision. On their view, this makes each of the AREA steps ‘more actionable, intuitive, and easy to remember’ [4].

Alternatively, Fudan 2023 used the STAR loop [5]. This approach involves four steps. First is Stimulation, involving identification of a problem and gathering of information. Second is Target, which involved finding the stakeholders. Third was Action, which involved establishing a dialogoue with relevant stakeholders. Finally, Review involved reflection of the feedback and integrating this to improve their project. They applied this framework to delineate each conservation with stakeholders or academics, which facilitated clear presentation of how these discussions informed their project. One team that we drew a lot of inspiration from, particularly in their regard to treating cultural concerns of Traditional Owners was UNSW 2021 [6]. They chose to employ the Double Diamond design process, created by the UK Design council. This consists of 5 key stages; Discover, Define, Develop, Deliver, and Evolve.

While many of the teams that we built our own approach around used a defined framework for human practices, this is not a necessity for a ‘good’ human practices approach. Several teams instead chose to identify core values for their team. For example, UCL 2022 settled on values including Team Diversity, Stakeholders with Diverse Backgrounds, and Education. Vilnius 2023 identified values of responsibility, reflection, sustainability, and quality-assurance, while Montpellier 2022 picked out concerns of farmers and good communication as core to their approach [7,8,9]. One advantage of the value-driven approach that we noted was that it may make human practices more accessible to individual teams compared to abstract frameworks, especially if these values are derived from group-brainstorming sessions.

Identification and interaction with stakeholders

The next question that we sought to answer in our analysis of approaches to human practices, was how previous teams went about engaging with stakeholders. Some teams adopted to use a framework specifically for this; Tec Chihuahua 2019 used a Triple Bottom Line (TBL) framework to look at the social, economical and environmental interactions their project had with stakeholders [10]. TBL has historically been used as an accounting framework, allowing business managers to balance financial with social and environmental measures [11]. We found this to be an innovative approach to stakeholder engagement by appropriating external frameworks.

Another project that stood out to us with Edinburgh UHAS Ghana 2022. Entering as a joint team, the value of collaboration was central to their project, and this is reflected in their approach to human practices, with an emphasis placed on establishing a network of relationships as a basis for sustainable stakeholder engagement [12].

Approaches to reflection

Another insight of our meta-analysis was that some teams decided to include a reflective tool in order to better reflect on stakeholder input. Stanford 2023 used the Atkins and Murphy Model of Reflection, an iterative process that focuses on five steps [13]. This is a 5-step model that involves the steps, Identify any learning, Awareness, Describe the situation, Analyse Feeling and Knowledge, and Evaluate Relevance of Knowledge [14]. The aim of this model is to support deeper level reflection and critical engagement with knowledge. By contrast, TU Eindhoven 2022 used the Gibbs Reflective Cycle, a six-module cyclical process that facilitates exploration of an experience [15]. This process involves, Description of the experience, Feelings and thoughts about the experience, Evaluation of the experience, Analysis to make sense of the situation, Conclusion about what you learned, and finally, an Action Plan for how you would deal with similar situations in the future [16]. We found that using such tools may help students and researchers undergo critical evaluation of the impact their science on stakeholders, in particular prompting non-superficial reflections. This helps to ensure that human practices is not simply a box to tick, but instead an integral part of the design cycle, feeding back and reflecting the values and interests of stakeholders.

Human practices and ethnography

This work formed a basis for the development of a standardized framework that we hope is useful for future iGEM teams who may be similarly daunted by the requirements of human practices. Ultimately, while a more systematic review is beyond the scope of our human practices this year, it provides a starting point for future research; science and the scientific process is an object of anthropological research, and ethnographic studies of laboratories have been conducted since the latter half of the 20th century in order to show how scientific products are ‘occasioned by the circumstances of their production’ [17]. More precisely, the context of scientific labs cannot be separated from the resultant scientific product. This is valuable to human practices; a deeper understanding of how science operates in laboratories and how scientific discussions occur even at the undergraduate level will aid in successful science communication, thereby fostering an increased alignment of scientific practice with important ethical, cultural, and social norms. We believe this to constitute a ‘good’ approach to human practices.

POLYP: Our Framework

From learning about prior iGEM team’s experiences with Human Practices, we brainstormed that we could making a significant contribution to future teams by trying to synthesize these differing approaches into a clear and facile framework that constitute a ‘good’ approach to human practices. We came up with POLYP, a stepwise and iterative process that provides a foundation for human practices.

Problem

Of course, any iGEM project should start with identifying the Problem at hand. This involves understanding the underlying issues that are contributing to the problem. Picking an issue to tackle is a daunting task in itself, and so a good place to start is by familiarising yourself with past iGEM projects – this can help show the scope of challenges people have addressed and the innovative ways used. It can be helpful to go through several rounds of pitching the problem getting feedback and answering any questions. In the context of CORA, we spent a long time in this step, looking at a wide range of areas before finally settling on the issue of coral bleaching

In this step you will likely start to develop potential synthetic biology solutions to the chosen problem. A good question to ask yourself is ‘where does our solution fit into the larger landscape of approaches’? While you probably can’t answer this question immediately, this will inform the future steps. Some key points to remember here are

• Think about what makes a good iGEM project – feasibility, impact, and innovation might be some criteria you consider.
• What are the current strategies being employed to tackle this issue. How would synthetic biology be an improvement on these?
• For the Primary and Secondary PI or any team mentors – the goal is not only to help the team choose a project, but also to prompt them to think about synthetic biology and research in general.

Orientate

Responsible research will recognize and respond to community perspectives. Consequently, any ‘good’ approach to human practices requires Orientating oneself towards the different stakeholders at play and to different core values that you or others might hold. Primary stakeholders are those who will be directly affected, but it is equally important to consider secondary stakeholders who may be indirectly affected by your project. You could also think about the Sustainable Development Goals (SDGs) and frame your research around these [18]. A good way to do this is to sit down with the team and have a few brainstorming sessions. Further, you may draw on the existing literature to reinforce your approach; have there been social science studies conducted into the application of synthetic biology to your chosen problem area? Some key points to remember here are

• Stakeholders’ interests will be many and varied – consider who might be impacted economically, culturally, socially, and environmentally by your project.
• Orientating isn’t exclusively about identifying stakeholders - an important part of the scientific process is the scientist! So, figuring out what values you have as a team can be a great way of getting everyone on the same page.

Learn

Importantly, the aim of human practices is not education nor outreach, but rather to generate a dialogue between researchers and the public. Thus, Learning from these stakeholders is an integral part of a ‘good’ approach to human practices. When looking at stakeholders in detail, it is likely that they will have divergent opinions on the research that you are doing. Further, outputs from stakeholders can result from many different methods of engagement, and using a mix of these can help to ensure that all voices are being heard. Some key hints to remember here are

• Learning is about engaging stakeholders, team members, and the general public in a discussion – it is important to be humble and listen, and to approach engagement in a multidisciplinary way.
• You don’t need to be an expert in science communication, the aim of human practices is to stimulate discussion, not to give a lecture.
• There are many tools that you can use here; surveys, consultation with experts, panels, field visits have all been employed by iGEM teams (to see how we went about it, please look at our ‘Survey’ section)
• Consider if there are any human research ethical concerns here – social science methods like surveys will sometimes require institutional approval, and will need to comply with iGEM’s human research policy [19]

Your thoughts

Next, we need to transform what we have learnt into integrated changes to our synthetic biology solution. While some previously used frameworks have used a singular step for this process, we chose to delineate this into two steps. The first invites critical reflection. As discussed in our meta-analysis, reflection on your thoughts about what you have learnt from stakeholder groups is a key tool in closing the loop between stakeholder discussions and the design cycle. The aim of this step is to promote reflexivity; a capacity for being able to consciously acknowledge one’s own beliefs, bias, and judgement systems [20]. Some key hints for this step:

• It is important to be critical here, human practices is not about affirming your existing project but instead about having open-ended discussions that will reinforce the utility of your solution for society.
• Indeed, you may find that your initial idea for a synthetic biology solution would not work/not be taken up by the intended end-users. This is okay – recognizing that here we aid future iterations and predictions of your design.
• One helpful tool that we implemented, inspired in part by takeaways from the Pacific Reef Collective Forum [21], was the usage of mind maps or drawing maps (see below). In particular, this tool may be effective for summarising larger group consultations such as those with community groups.

Predict

Flowing on from critical reflection, our final step of the framework expands on reflection and transforms it into action. Having now brought attention to certain deficits within your project or certain important community values or interests, a ‘good’ approach to human practices should then Predict the interplay between the science and these considerations. The overall aim here is to diminish the disconnect between the general public and science by showing that you care about their perspective. It is natural in this stage to realise that there might be problems with your current approach or adjusted approach, and thus the framework intuitively takes you back to the first step, entailing a continuing and evolving approach to engage with human practices. Some key points to remember here are:

• Often, we cannot immediately predict all the implications of our synthetic biology solution. Thus, a tool like a Risk Matrix, which assess risks and determines a risk rating based on different likelihood and consequence criteria [22], might be useful here.
• Human Practices is not a one-time thing, but rather a dynamic and evolving process that unfolds concurrently with R&D. So, it can be good to predict how public perceptions might change, and what future engagement with the public might look like.

Overall, we think that this framework represents a good and responsible approach to human practices, by giving greater attention to some concerns present in social science research. It also ameliorates some limitations in human practice tools that past teams have used; as opposed to some of the more abstract frameworks that are non-specific and have been appropriated to synthetic biology, POLYP was written by and for synthetic biologists. Hopefully this is reflected in the value of the framework, and that future teams find it both readily accessible and useful towards their own human-centric approach to human practices.

Consultation with Dr. Line Bay

Dr. Line Bay is the Research Director of the Reef Recovery, Adaptation and Restoration Program (RRAP) and at the Australian institute of Marine Science (AIMS). Here, she leads a team focussed on understanding coral reef ecology and evolution. She is also an enthusiastic science communicator and educator with an interested of translating scientific knowledge to natural resource managers, policy makers and the general public.

Through our consultation with Line, we learnt about some of the challenges and value of science communication and engagement with stakeholders. In particular her comments on the challenges that RRAP have faced with the regulatory framework of the Marine Park Authority (MPA) led us to consider and refine the future phases of our project. We decided to start looking at the implementation of our project in more depth, and made us consider the usage of a kill switch in order to mitigate risks associated with small-scale in situ experiments.

She also prompted us to think about the end-users of CORA. While we had already identified governmental agencies and non-government organizations (NGOs) as potential end-users, she pointed out that there is no industry taking responsibility for reef conservation, and emphasized the role that local communities should play. To read more about our proposed full package implementation of CORA, please see our future directions section.

Survey

Designing a solution that is responsible and socially beneficial requires understanding the opinions and values of wider society. One of the most important questions in human practices is how could we, as scientists, best engage with society’s concerns and ensure that their values are incorporated into our proposed solution?

Here, we chose a human-centric approach in order to obtain a diversity of perspectives from the public, and using these perspectives to inform the phases of development of our project. Dr Dan Santos of the ANU Centre for the Public Awareness of Science suggested that we should approach the problem as an exercise in social sciences, and we worked in collaboration with him to write and submit an application to the ANU Human Research Ethics Committee to undergo a survey of communities local to the reef.

Our survey built upon existing work by CSIRO in their ‘Public perceptions of using synthetic biology to restore the Great Barrier Reef’ report and work by the 2021 UNSW iGEM team [6, 23]. Following on from these results, key questions that our survey intended to address included;

• Whether participant was aware of synthetic biology and extent of their knowledge
• Consideration of ‘willingness to interact with’, ‘how bothered’ would participants be if technology was introduced in their local area (i.e. proximal behavioral intentions)
• Their willingness to support research with various synthetic biology techniques (removing gene, adding gene from same species, transgenics, changes to existing gene)
• What role do they think local communities should play in devising and implementing conservation policy.

These results would play a key role in dictating the implementation and subsequent design cycles of CORA, ensuring that valuable public insights are captured in our design. Unfortunately, the ANU Human Research Ethics Committee were still deliberating on our application at the time of the wiki freeze, and thus, we were not able to share our survey with the stakeholder groups of interests. However, going through this process gave us valuable insights into some of the considerations relevant to human and social science research.