DESCRIPTION
Background
In today's fast-paced society, there is a shared yearning for respite from the monotony and a desire to find solace in nature. The connection toward the soothing embrace of natural environments, the tranquil forests, awe-inspiring mountains, and captivating ocean has become integral facets of the human experience, profoundly influencing our existence. Especially, the exquisite balance of marine ecosystems, characterized by intricate food chains and finely tuned biodiversity, is a masterpiece of natural harmony. However, under the sea, this delicate equilibrium is under a grave threat of irreversible disruption. 71% of Earth is made up of water[1], teeming with vibrant life, but this life is now threatened by the irreversible destruction of coral reefs, which support more than 25% of marine life [2]. Beyond their ecological significance, their economic importance is substantial. In 2018, The UN Environment Programme stated that coral reefs contribute valuable resources and services to society, with an estimated annual worth of $375 billion [3]. Renowned for their breathtaking vibrancy and the intricate tapestry of life they sustain, coral reefs are experiencing a precipitous decline. Without urgent intervention, these magnificent ecosystems, the very heartbeats of our planet, may vanish completely as soon as 2050.
Graph 1. The economic benefits provided by ecosystem services for coral reef ecosystems. Values, displayed logarithmically in US$/ha/year, represent both the average and maximum values. [4]
Corals are complex organisms that survive on the synbiotic cooperation between a photosynthetic algae and multiple coral polyps; without the algae to provide them with energy, corals cannot survive. Coral bleaching is a phenomenon that occurs when heat stress from rising temperatures forces the photosynthetic algae to be expelled from coral surfaces. This loss of the coral’s primary energy source makes the coral vulnerable to foreign threats such as coral tissue loss diseases, leading to a significant decline in coral populations. These coral disasters have already been witnessed in the Caribbean and Australia. Beyond bleaching, carbon dioxide absorbed into the ocean from the atmosphere has already begun to reduce calcification rates (coral growth) in reef-building and reef-associated organisms by altering seawater chemistry through decreases in pH. [5]
Current solutions addressing coral diseases involve various strategies. However, the root cause, global warming, still cannot be easily resolved. Increased sea temperatures and ocean acidification caused by climate change exacerbate coral diseases.
Current Solutions to Coral Conservation
One of the predominant efforts in combating reef decline is coral restoration programs. These restoration programs, such as coral nurseries and outplanting projects, focus on propagating and transplanting disease-resistant coral species in the ocean. But there is a problem: corals grow too slowly, growing only 0.3 to 2 cm a year. To put that into perspective, it takes approximately 10,000 years for a group of coral larvae to grow into a full coral [6]. 90% of corals are predicted to die in the next two decades, and we simply cannot outgrow the rapid rates of coral deaths [7]. This leads us to pursuing an alternative solution: conservation.
One of the most common solutions at the moment for conservation is antibiotic pastes. Antibiotic pastes are smeared onto the coral surface and can provide a rapid response to coral disease outbreaks, making it efficient when trying to prevent the spread of disease within a particular reef ecosystem. However, the use of antibiotics carries significant concerns for the environment; one of them being the development of resistant coral strains due to over or improper use of antibiotics. Additionally, antibiotics lack specificity; they cannot distinguish between harmful and beneficial bacteria. This indiscrimination could kill off good bacteria that play crucial roles in coral health.
A Glimpse of Hope
Recently, probiotics have been found to address the challenges faced by corals in response to their changing environmental conditions [8]. These beneficial microorganisms, known as BMCs, have displayed their capacity to enhance coral well-being through various mechanisms, including building resistance against diseases. Recent studies indicate that probiotics offer a rapid and effective natural approach for corals to adapt and flourish amidst shifting environmental circumstances [8]. In particular, researchers are actively investigating the efficacy of probiotics in combating diseases such as SCTLD through field trials conducted within reef ecosystems [9]. Stony coral tissue loss disease (SCTLD) is a highly lethal coral disease that was first reported off the coast of Florida in 2014 and has since spread rapidly throughout the Caribbean. The disease affects over 20 coral species and is now present on reefs in 18 countries and territories [10]. The idea of boosting advantageous indigenous microbes within the microbiome—a mutualistic entity composed of host organisms together with associated microbial communities—has gained considerable traction among coral scientists exploring the impact of probiotics on mitigating adverse effects arising from environmental changes. Despite their benefits, delivering probiotics to coral reefs still remains a challenge.
Probiotic Beneficial Mechanism
Pseudoalteromonas sp. Strain McH1-7 [9][11]
  • Treats Stony Coral Tissue Loss Disease (SCTLD) directly
  • Acts as a prophylactic
Pseudoalteromonas luteoviolacea [12]
  • Stimulates coral metamorphosis
Pseudoalteromonas sp., Halomonas taeanensis and Cobetia marina-related Species Strains [13]
  • Promotes resistance towards coral bleaching
Table 1. Beneficial probiotics to corals and their beneficial mechanism
Current Probiotic Delivery Systems & Their Issues
One of the most common ways to deliver the probiotics is through plastic bags that are implemented to trap probiotics in the coral’s immediate vicinity; this helps corals absorb probiotics more efficiently. However, there are still many issues with this delivery system:

  1. Low scalability Requires a large amount of logistical effort to put and remove plastic bags.
  2. Low Absorption: Probiotics only in proximity to the coral, but not attached to the surface, causing low absorption efficiency.
  3. Create harmful coral micro-environments: The plastic blocks sunlight, leading to low-oxygen conditions that can promote the growth of disease-causing bacteria. Research shows that corals without any plastic wrapping had a 4% chance of being diseased, but the presence of plastic raised the risk to 89% [14].
  4. Environmental Harm: Having plastics in the ocean causes pollution – plastics break down into microplastics, fishes could consume them, and more.
Another delivery mechanism currently under research is using rotifers. Rotifers are multicellular animals with body cavities that are partially lined by mesoderm. These organisms have specialized organ systems and a complete digestive tract that includes both a mouth and anus [15]. The method of utilizing rotifers involves feeding rotifers probiotics then feeding the rotifers to the corals. As the corals ingest the rotifers, they also absorb the probiotics in the rotifers. However, this method also has its issues:

  1. Low scalability: Long preparation time, have to grow rotifers with the specific probiotics that needs to be delivered.
  2. Compatibility concerns: Not all probiotics will be suitable for rotifer consumption, and even if they are, their viability and effectiveness may be compromised during the process, as it's hard to maintain the viability of the probiotics through the gut of the rotifers and during the transit to the coral's environment. Harsh environmental conditions or exposure to stomach acids in the rotifer's digestive system might reduce the effectiveness of the probiotics when they reach the corals.
Our Solution: Cure-All Reef
Coral reefs are facing an unprecedented threat. The beating heart of our world is getting closer and closer to being irreversibly disrupted. There hasn’t been an efficacious and efficient method, despite active research by respective experts. Thus, our project aims to increase probiotic retention on corals, which can strengthen the immune system of corals. To help our dying corals, our Cure-All Reef team proposed an innovative and sustainable approach to probiotic delivery - Cure-All Eats. Our plan involves engineering SAR11 bacteria, a common food source of corals, to secrete biofilm to act as a temporary glue. By co-culturing our special blend of probiotics and engineered SAR11, the probiotic will be able to attach onto the coral mucus and integrate into the coral microbiome. However, through the comprehensive assessment of all pertinent factors, we decided to select E. coli as the preferred experimental chassis for our delivery system with the intention to reserve SAR11 for future use. E. coli is deemed eminently suitable for our experimental framework due to its inherent simplicity and the facility with which it can be cultivated and subjected to laboratory scrutiny. The utilization of E. coli in validating our conceptual approach also confers the advantage of enhancing the experiment’s outcome clarity, obviating the need for simulations involving oceanic and seawater conditions.
The release of GMOs into the wild is considered harmful to the environment and is subject to strict regulations and ethical considerations. Understanding that, our team designed a system that serves to prevent potential environmental harm.
Figure 1. A big picture of the problem and our solution.
Biofilm
        Two crucial gene clusters play a role in the synthesis and control of biofilm. The first, the Csg operon, contains the genes necessary for producing curli protein [16] necessary for biofilm formation. The second, the metJ regulon, functions as a time-based and quorum-sensing regulator that represses biofilm production by inhibiting csg biofilm forming activity.
We designed two constructs to host these two gene clusters and made sure they can work together for biofilm production and regulation, named the csg construct and the metJ construct, respectively. This regulatory mechanism operates through interactions between the met operator within the Csg construct and the MetJ regulatory protein produced by the metJ construct; when the MetJ regulatory protein binds to the met operator, it represses the csg production, leading to the reduction of biofilm production. To regulate the production of MetJ, Rail (located in the MetJ construct) codes for a synthase that produces an autoinducer called AHL (Acyl-homoserine lactone). AHL concentration mediates bacteria quorum sensing and regulates metJ production; higher AHL concentration leads to more metJ production and therefore less biofilm production [17].
In order to ensure that our biofilm grows sufficiently to attach probiotics to the coral mucus before the MetJ regulatory protein decreases its production rate, we utilized a strong promoter in the Csg construct, while using a weak promoter in the MetJ construct.
For more information about our experimentation, please visit the experiments page [18].
Figure 2. Biobrick diagram of our Csg construct
Figure 3. Biobrick diagram of our MetJ construct
Coral-Localization Kill Switch System [19][20]
        To restrict the presence of bacteria exclusively around targeted corals, our team has developed a coral-localization kill switch system inspired by the toxin-antitoxin system designed by Johns Hopkins 2011 iGEM Team [21]. This system is based on population density with the utilization of the quorum-sensing mechanism. Antitoxins are constantly produced. At high density, AHL (produced by our metJ construct) suppresses toxin production while MazE proteins (anti-toxin) bind to the remaining MazF proteins(toxin), preventing MazF to exert its toxicity through recognizing and cleaving mRNAs. When factors such as ocean currents and marine life intervention disperses our engineered bacteria away from the biofilm and its targeted coral, lowered AHL concentrations cause production of toxins, triggering bacteria death. This self-destructive biosafety mechanism was approved by Dr. Tang Sen-Lin and Professor David Bourne from in-person and online meetings held during the summer.
For more information about our experimentation, please visit the experiments page [22].
Figure 4. Biobrick diagram of our Biosafety construct
Implementation
To actualize the use of our probiotics for coral conservation, we've designed a spray mechanism paired with a robot that can allow probiotics to be delivered autonomously underwater. We created a pneumatic spray mechanism to deliver atomized droplets of solution including our engineered bacteria and probiotic.
We hope that through our development of Cure-All Eats, it can save more corals from dying. After all, saving corals is not just saving a species, it’s about saving a whole ecosystem. For more information, please visit our hardware page [23].
References
[1] How Much Water is There on Earth? | U.S. Geological Survey. (2018, June 6). https://www.usgs.gov/special-topics/water-science-school/science/how-much-water-there-earth
[2] Environment, U. (2021). Status of Coral Reefs of the World 2020. UNEP - UN Environment Programme. https://www.unep.org/resources/status-coral-reefs-world-2020
[3] Peixoto, R. S., Rosado, P. M., Leite, D. L., Alexandre Soares Rosado, & Bourne, D. G. (2017). Beneficial Microorganisms for Corals (BMC): Proposed Mechanisms for Coral Health and Resilience. Frontiers in Microbiology, 8. https://www.researchgate.net/publication/332083911_Adapting_to_extreme_environm
ents_can_coral_reefs_adapt_to_climate_change
[4] James, M. (2019, March 29). Adapting to extreme environments: can coral reefs adapt to climate change? ResearchGate; Portland Press. https://www.researchgate.net/publication/332083911_Adapting_to_extreme_environm
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[5] US. (2023). How does climate change affect coral reefs? Noaa.gov. https://oceanservice.noaa.gov/facts/coralreef-climate.html
[6] ‌US. (2023). How Do Coral Reefs Form: Corals Tutorial. Noaa.gov. https://oceanservice.noaa.gov/education/tutorial_corals/coral04_reefs.html#:~:text=W
ith%20growth%20rates%20of%200.3,30%2C000%2C000%20years%20to%20fully%20form [7] Davidson, J. (2020, February 20). Coral Reefs Could Be Completely Lost to the Climate Crisis by 2100, New Study Finds. EcoWatch; EcoWatch. https://www.ecowatch.com/coral-reefs-climate-crisis-predictions-2645201373.html
[8] Peixoto, R. S., Rosado, P. M., Leite, D. L., Alexandre Soares Rosado, & Bourne, D. G. (2017). Beneficial Microorganisms for Corals (BMC): Proposed Mechanisms for Coral Health and Resilience. Frontiers in Microbiology, 8. https://pubmed.ncbi.nlm.nih.gov/28326066/
[9] Ushijima, B., Gunasekera, S. P., Meyer, J., Tittl, J., Pitts, K. A., Thompson, S., Sneed, J. M., Ding, Y., Chen, M., L Jay Houk, Aeby, G. S., Häse, C. C., & Paul, V. J. (2023). Chemical and genomic characterization of a potential probiotic treatment for stony coral tissue loss disease. Communications Biology, 6(1). https://doi.org/10.1038/s42003-023-04590-y
[10] Ushijima, B., Gunasekera, S. P., Meyer, J., Tittl, J., Pitts, K. A., Thompson, S., Sneed, J. M., Ding, Y., Chen, M., L Jay Houk, Aeby, G. S., Häse, C. C., & Paul, V. J. (2023). Chemical and genomic characterization of a potential probiotic treatment for stony coral tissue loss disease. Communications Biology, 6(1). https://doi.org/10.1038/s42003-023-04590-y
[11] Stony Coral Tissue Loss Disease (SCTLD) - Coral Disease & Health Consortium. (2023, January 23). Coral Disease & Health Consortium. https://www.academia.edu/25167010/Co_ordination_of_quorum_sensing_regulation_i
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[12] Coral Disease Outbreak - AGRRA. (2023, August 29). AGRRA. https://www.agrra.org/coral-disease-outbreak/
[13] Alker, A. T., Delherbe, N., Purdy, T., Moore, B. S., & Shikuma, N. J. (2020). Genetic examination of the marine bacterium Pseudoalteromonas luteoviolacea and effects of its metamorphosis‐inducing factors. Environmental Microbiology, 22(11), 4689–4701. https://doi.org/10.1111/1462-2920.15211
[14] Rosado, P. M., Leite, D. L., Duarte, G., Chaloub, R. M., Guillaume Jospin, Nunes, U., Saraiva, J., Dini‐Andreote, F., Eisen, J. A., Bourne, D. G., & Peixoto, R. S. (2018). Marine probiotics: increasing coral resistance to bleaching through microbiome manipulation. The ISME Journal, 13(4), 921–936. https://doi.org/10.1038/s41396-018-0323-6
[15] Introduction to the Rotifera. (2023). Berkeley.edu. https://ucmp.berkeley.edu/phyla/rotifera/rotifera.html
[16] Gene Expression Regulation by the Curli Activator CsgD Protein: Modulation of Cellulose Biosynthesis and Control of Negative Determinants for Microbial Adhesion | Journal of Bacteriology. (2023). Journal of Bacteriology. https://journals.asm.org/doi/10.1128/jb.188.6.2027-2037.2006
[17] The cin and rai Quorum-Sensing Regulatory Systems in Rhizobium leguminosarum Are Coordinated by ExpR and CinS, a Small Regulatory Protein Coexpressed with CinI | Journal of Bacteriology. (2023). Journal of Bacteriology. https://journals.asm.org/doi/10.1128/jb.01650-08
[18] Experiments | GEMS-Taiwan - iGEM 2023. (2023). Igem.wiki. https://2023.igem.wiki/gems-taiwan/experiments
[19] Susanne, Majerczak, D. R., & Coplin, D. L. (1998). A negative regulator mediates quorum-sensing control of exopolysaccharide production in Pantoea stewartii subsp. stewartii. Proceedings of the National Academy of Sciences of the United States of America, 95(13), 7687–7692. https://doi.org/10.1073/pnas.95.13.7687
[20] Team:Hopkins - 2021.igem.org. (2021). Igem.org. https://2021.igem.org/Team:Hopkins
[21] Boss, L., & Kędzierska, B. (2023). Bacterial Toxin-Antitoxin Systems’ Cross-Interactions—Implications for Practical Use in Medicine and Biotechnology. Toxins, 15(6), 380–380. https://doi.org/10.3390/toxins15060380
[22] Experiments | GEMS-Taiwan - iGEM 2023. (2023). Igem.wiki. https://2023.igem.wiki/gems-taiwan/experiments
[23] Implementation | GEMS-Taiwan - iGEM 2023. (2023). Igem.wiki. https://ucmp.berkeley.edu/phyla/rotifera/rotifera.html