Team:UNSW Australia/Poster

PROTECC CORAL

TEAM UNSW AUSTRALIA 2020

PROTECC Coral (Prevent Reactive Oxygen and Thermal Extreme Caused Carking)


Authors:


Annabelle Beach, Annabelle Lee, Caitlin Ramsay, Chelsea Liang, Cornelius Bong, Deborah Chandra, Farnaz Sedghidiznab, Gabrielle Milet, Jack Robbers, Jason Lin, Kelley Gao, Kelly Varianne, Samuel Humphrey, Sakthirupini Ramamurthy, Sayali Gore, Zelun Li
All affiliated with UNSW.

Abstract:


Home to three-quarters of the world’s coral population, Australia’s Great Barrier Reef (GBR) is experiencing its third bleaching event in five years. With increasing ocean temperatures, coral reefs and the ecosystems that depend on them are at risk of serious, irreversible damage. Coral bleaching is a result of heat-induced oxidative stress, which triggers the ejection of corals’ microalgal-symbiont Symbiodinium spp. PROTECC Coral aims to improve the heat tolerance of coral by introducing small heat shock proteins alongside a glutathione recycling enzyme system into Symbiodinium spp., in order to reduce cellular stress. By engaging in conversations with various stakeholders (tourism industries, local councils, bioprospecting practices, commercial and recreational fishing and the GBR’s traditional owners) and integrating their advice, our team hopes to contribute to a worldwide conservation effort that enables future generations, both Australian and non-Australian alike, to enjoy the GBR in its entirety.

INTRODUCTION & INSPIRATION

INTRODUCTION & INSPIRATION



Why We Care About Coral Bleaching


Australia’s global identity is hallmarked by the Great Barrier Reef, a biologically diverse ecosystem located off the coast of Queensland, Australia. Its coral reef is the largest in the world, containing over 600 coral species, and accounting for three-quarters of the world’s coral population. (1)

The UNSW iGEM team is made up of young people who call Australia home, and for whom the GBR is a national treasure. The reef is steeped in a history and culture that runs deep in our communities, with approximately 70 Traditional Owner groups’ sacred connections spanning back tens of thousands of years. (2) For many of us, the reef has taught us about the deep connection between human and land, and the vast complexities found within ecosystems. It has been a muse for art and stories; provided thousands of Australians identity and livelihood; acted as a holiday destination for many Australian and non-Australian families; and imparted inspiration for thousands of school-science projects. It has left its mark on our parents, our grandparents and our great-grandparents, and has ingrained in us a passion to carry this legacy on to future generations.

With all its history in our foundations, we face our dying GBR with one primal instinct: to want to protect our home. We believe ignoring it would be a great disservice to the generations of Aboriginal and Torres Strait Islander peoples who spent thousands of years protecting it before us. So, when given an opportunity to focus on one problem this year, we could think no further than the one happening in our own backyard.

Our Inspiration


As we face the problem of coral bleaching, we draw on inspiration from many people around us.

Firstly, we are inspired by the immense strength the Australian people have shown in the face of lost loved-ones, homes and livelihoods; a result of the effects of climate change.

Secondly, we are inspired by those who are contributing to a worldwide effort to conserve natural environments with advanced biotechnology. One of our sponsors, Revive & Restore (R&R), has implemented a ‘genetic rescue toolkit’, which features a crossover of genetic rescue, a standard conservation strategy, with advanced biotechnologies, such as genetic engineering and gene editing tools. (3) Seeing R&R navigate this crossover so gracefully has pushed us to imagine the possibilities of our own project, and explore the idea of this crossover in the context of coral bleaching. Thirdly, we are deeply inspired by those who have been contributing to the global effort to save coral reefs for decades. Coral bleaching has been detrimental to countless marine ecosystems worldwide, and the severity of the coral bleaching problem cannot be understated. The UNSW iGEM 2020 Team is simultaneously proud and humbled to be contributing to a larger, global effort towards a long-term, practicable solution for coral bleaching. We are passionate about working towards our common goal: to preserve and restore coral reefs so future generations can enjoy them in their magnificent entirety.

References

  1. Great Barrier Reef Marine Park Authority [Internet]. Reef facts. [place unknown]: Australian Government; 2020 [cited 2020 Oct 27]. Available from http://www.gbrmpa.gov.au/the-reef/reef-facts
  2. Great Barrier Reef Marine Park Authority [Internet]. Traditional Owners. [place unknown]: Australian Government; 2020 [cited 2020 Oct 21]. Available from http://www.gbrmpa.gov.au/our-partners/traditional-owners
  3. Revive and Restore [Internet]. What we do. [place unknown]: Revive and Restore; 2020 [cited 2020 Oct 21]. Available from https://reviverestore.org/what-we-do/

THE PROBLEM

Coral Bleaching & Our IHP Approach

THE PROBLEM


The Problem: Coral Bleaching


Reef building coral are complex, interconnected structures composed of polyps, each containing symbiotic microalgae such as Symbiodinium spp. within their tissues. These microalgae generate nutrients necessary for survival and establish a close relationship with their coral hosts. (1) Climate change causes stressful conditions, such as rising ocean temperatures, resulting in thermally induced protein denaturation and oxidative stress. The production of damaging reactive oxygen species (ROS) by the Symbiodinium triggers expulsion from the coral, resulting in coral starvation and eventual death. (2, 3) As the microalgae leaves the coral polyp, the white skeleton remains, representing what we know as the bleached-white expanse that already blankets more than 60% of the Great Barrier Reef. (4, 5)

Our Human Centred Approach To Coral Bleaching



The GBR, a hallmark of Australia’s national identity, is currently experiencing its third coral bleaching event in just five short years.The impacts of climate change, such as rising ocean temperatures, are devastating on natural environments. We have watched as Australian communities, industries and environments have been directly affected by corals bleached white. However, the problem of coral bleaching exists not only in the devastation of a natural and biodiverse ecosystem. Coral bleaching causes far-reaching problems for the people and societies who are deeply connected to, and depend on a healthy coral reef. This was the foundation of our team’s human-centred approach.

Our initial consultations with conservationists, ethicists and social scientists helped establish our understanding of the social landscape of synthetic biology solutions in natural environments.Then, we identified five key stakeholders; relationships that would be sustained through and ultimately drive the development of our solution to coral bleaching. To each of these stakeholders, coral bleaching presents a real and serious problem.

  1. Traditional Owners, who possess sacred cultural connection to the land, including the Great Barrier Reef region, face a degradation of history and culture.
  2. The rich biodiversity of the GBR, encompassing coral species, as well as the marine life dependent on coral reefs, faces loss.
  3. The bioprospecting industry faces a depletion of medical and pharmaceutical resources that may be used to treat presently incurable diseases.
  4. The integrity of coastlines that are vulnerable to wave action and storms without the protection of coral reefs.
  5. Tourism and fishing industries face major economic damage, as do the livelihoods of more than 69,000 Australians.

    Through ongoing discussions with stakeholders, we aimed to build bilateral conversations to lessen the disconnect between science and society. These conversations helped us ensure that our project remained 'human-centred' and 'values-driven'. Only when people and science exist symbiotically, do solutions which are good and responsible for the world arise.

    The GBR carries much national and emotional weight for Australians, whether or not they are directly impacted by coral bleaching. The preservation of a healthy coral reef is symbolic in conversations about the effects of climate change and the potential of synthetic biology solutions. The overarching problem of climate change may continue to persist. However, the powerful potential of synthetic biology solutions to preserve the Australian and global coral reefs, can help ensure that future generations can enjoy them in their entirety.

References

  1. Roth MS. The engine of the reef: photobiology of the coral–algal symbiosis. Front Microbiol [Internet]. 2014 [cited 2020 Oct 27];5. Available from https://www.frontiersin.org/articles/10.3389/fmicb.2014.00422/full
  2. Vidal-Dupiol J, Adjeroud M, Roger E, Foure L, Duval D, Mone Y, et al. Coral bleaching under thermal stress: putative involvement of host/symbiont recognition mechanisms. BMC Physiol. 2009 Aug 4;9(1):14.
  3. Lesser MP. Coral Bleaching: Causes and Mechanisms. In: Dubinsky Z, Stambler N, editors. Coral Reefs: An Ecosystem in Transition [Internet]. Dordrecht: Springer Netherlands; 2011 [cited 2020 Oct 27]. p. 405–19. Available from https://doi.org/10.1007/978-94-007-0114-4_23
  4. Long-term shifts in the colony size structure of coral populations along the Great Barrier Reef | Proceedings of the Royal Society B: Biological Sciences [Internet]. [cited 2020 Oct 27]. Available from https://royalsocietypublishing.org/doi/10.1098/rspb.2020.1432
  5. Bieri T, Onishi M, Xiang T, Grossman AR, Pringle JR. Relative Contributions of Various Cellular Mechanisms to Loss of Algae during Cnidarian Bleaching. PLOS ONE. 2016 Apr 27;11(4):e0152693.
  6. Great Barrier Reef suffers third mass bleaching in five years. BBC News [Internet]. 2020 Mar 26 [cited 2020 Oct 27]. Available from https://www.bbc.com/news/world-australia-52043554

PHASE I: DESIGNING OUR SOLUTION

Engineering Thermo-tolerant Symbiotic Microalgae

PHASE I: DESIGNING OUR SOLUTION

The aim of our project is to use synthetic biology to engineer more thermotolerant Symbiodinium hosts. To achieve this, two approaches have been developed by our team that both involve the introduction of genes via plasmid transformation. In order for our constructs to be responsive to coral bleaching, these genes will be under the control of the YAP1 ROS inducible promoter.

The first approach is on the enzymatic level with the incorporation of glutathione (GSH); a ubiquitous primary antioxidant essential for neutralising ROS into less harmful products. It achieves this in its reduced thiol form by donating available electrons to ROS and thus preventing the oxidation of biological molecules. (1) The bifunctional glutathione synthetase gene (gshF) was selected due to its lack of a complex inhibition system, enabling greater GSH production. (2) To recycle oxidised GSH back into its reduced form, glutathione reductase will also be included. In this way, an efficient glutathione system can be introduced into our system to prevent ROS accumulation.

The second approach in our project is the addition of small heat shock proteins (sHSP). These are protein chaperones that prevent protein misfolding from heat stress through its holdase activity. By binding to the hydrophobic core of denaturing proteins, sHSPs prevents their solubilisation and hence, interaction with other molecules. In this way, insoluble protein aggregates and its associated cellular stress can be avoided. (3)

There are various features of sHSP that make it ideal to introduce into Symbiodinium, prevent thermal denaturation and supplement the glutathione system. For one, its non-specificity enables sHSP to act on a large library of substrates. The ATP-independent nature of sHSP reactions is greatly beneficial in the context of oxidative stress as overburdened mitochondria inadvertently produce more ROS. (4) Furthermore, sHSP recruit other ATP-dependent chaperones to increase refolding by more than 80%. (5) Hence, for these reasons, sHSPs were a large focus in our solution.

For our project, HSP22 from Chlamydomonas reinhardtii was chosen due to its functionality at temperatures over 42°C; well above the sea surface temperatures of 30°C at Great Barrier Reef where coral bleaching occurs. (6, 7) More specifically, HSP22E and HSP22F were selected as these proteins work in conjunction with each other to target misfolding in the chloroplast; a major source of ROS leakage. (4)

References

  1. Wang Y, Li H, Li T, Du X, Zhang X, Guo T, Kong J. Glutathione biosynthesis is essential for antioxidant and anti-inflammatory effects of Streptococcus thermophilus. International Dairy Journal. 2019 Feb 1;89:31-6.
  2. Li W, Li Z, Yang J, Ye Q. Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli. Journal of biotechnology. 2011 Jul 20;154(4):261-8.
  3. Mogk A, Ruger-Herreros C, Bukau B. Cellular functions and mechanisms of action of small heat shock proteins. Annual review of microbiology. 2019 Sep 8;73:89-110.
  4. Kobayashi Y, Harada N, Nishimura Y, Saito T, Nakamura M, Fujiwara T, Kuroiwa T, Misumi O. Algae sense exact temperatures: small heat shock proteins are expressed at the survival threshold temperature in Cyanidioschyzon merolae and Chlamydomonas reinhardtii. Genome biology and evolution. 2014 Oct 1;6(10):2731-40.
  5. Lee GJ, Vierling E. A small heat shock protein cooperates with heat shock protein 70 systems to reactivate a heat-denatured protein. Plant Physiology. 2000 Jan 1;122(1):189-98.
  6. Great Barrier Reef Tours. 2020. Annual Great Barrier Reef Weather Overview. [online] [Accessed 22 October 2020]. Available from https://greatbarrierreeftourscairns.com.au/blog/annual-great-barrier-reef-weather-overview/#:~:text=Temperatures%20are%20pretty%20steady%20throughout,2010mm%20falling%20during%20the%20year.
  7. Jackson R, Gabric A, Cropp R. Effects of ocean warming and coral bleaching on aerosol emissions in the Great Barrier Reef, Australia. Scientific reports. 2018 Sep 19;8(1):1-1.

EXPERIMENT

EXPERIMENT

Our experimental work aimed at characterising the chaperone activity of novel small heat shock proteins; HSP22E and HSP22F. This was done by assembly of the gene construct in the pET-19b plasmid and transforming the plasmids in E. coli chassis. E. coli was our chosen chassis due to their high growth rate and transformation efficiency. This was beneficial for producing large amounts of protein for characterisation, especially given lab access limitations due to COVID-19. Induction of HSP22E and 22F expression was followed by His-tag protein purification and assessing the activity of the chaperones in preventing protein aggregation. It was found the presence of the HSPs greatly reduced aggregation when compared to samples without the chaperones. This suggests HSP22E and 22F could be effective in preventing irreversible heat damage within Symbiodinium.

Figure 1: HSP22E (●) and HSP22F (Δ) are shown to decrease heat-induced aggregation of citrate synthase (CS). Aggregation of CS was quantified by changes in absorbance at 500 nm over time.

Additionally, we conducted further characterisation by investigating their intermolecular interactions, involved in forming higher-order complexes. It became apparent that heat shock proteins 22E and 22F formed dimers with themselves through disulfide bonds. This was deduced as proteins were visualised through SDS-PAGE at both 37 kDa, 70 kDa and 140 kDa in denaturing conditions, however in the presence of a reducing agent (DTT), disulfide bonds ceased to form and HSP were mostly present in their monomeric form at 37 kDa. Further visualisation in their native state indicated formation of larger order complexes at 720 kDa and higher.

Figure 2: SDS and Native PAGE A) SDS-PAGE. The lowest boxes indicate monomeric HSP at 37 kDa, whilst higher boxes contain complexes at around 70 and 140 kDa. The last 3 samples were incubated with DDT. B) Native PAGE with higher order complexes evident at top of the gel above 720 kDa.

MODELLING

MODELLING

Structural



We utilised fold recognition template modelling with the i-TASSER server to impose a reasonable 3D structure onto a sequence of peptides. Unfortunately, scientific literature could only say generally that heat shock proteins form dimers and then large oligomers but not tell us whether they are functional as a dimer or an oligomer. This was the central question we wanted to ask. After docking a HSP22E and HSP22F together to form a heterodimer, we conducted de novo loop remodelling to remove atom clashes from our dimer model. This was assessed for convergence on a structure (Figure 1) and a 12-unit oligomer was constructed from these dimers (Figure 2). In Phase I, foundational structural models were created for further molecular dynamics to better understand the functional qualities of algae heat shock proteins.

Figure 1: The HSP22E/F dimer was simulated for 275ns to observe the changes in conformation of the protein. Simulation was done with gromacs with the CHARMM27 force field. The monomers in the dimer didn’t float away from each and for the radius of gyration decreased and appeared to stabilise which gave us greater confidence that this might be how these dimers dock together.

Figure 2: 12-unit oligomer model created with another eukaryotic heat shock protein complex as a template. Dimers were aligned in pymol against the template. After refinement and removal of overlaps, this model would be the basis for an oligomer model of the HSP22 complex.

Mathematical



Heat shock responses are ubiquitous in living organisms as all environments have temperature fluctuations and a need to mitigate oxidative stress. Mathematical models of the new engineered system were compared to a model of the wild type system to predict the biochemical changes on the level of the cell. The comparison between the 2 models were aimed at analyzing the effectiveness of our proposed engineering solution in a quantitative measure. We consulted Mark Tanaka, who helped us understand the difference between stochastic and deterministic versions of these models, we ultimately implemented stochastic models using the PySB library. The plot of various substrate levels in a wild type Symbiodinium model(Figure 3) and in our engineered Symbiodinium model(Figure 4) at an elevated temperature is compared.

Figure 3: The Natural Protein level plummeted in our simulation time frame of a 100 seconds, with noticeable ROS level surge

.

Figure 4: The Natural Protein level is maintained at a relatively healthy level, even as the ROS concentration is suppressed heavily by rapid glutathione production.

From the graph we can see that by introducing sHSP and Glutathione the level of Natural Proteins is conserved at a relatively stable range.

PHASE II: FUTURE DIRECTIONS

Biosafety & Ex-Situ Testing

PHASE II: FUTURE DIRECTIONS

In Phase II, we aim to implement our adapted genetic constructs into Symbiodinium goreui for introduction into coral.

One initial consideration, as with any proposed release of GMOs, is the biosafety implication of our solution. To limit risks surrounding invasion of the modified Symbiodinium outside of its intended environment, we have designed a toxin/antitoxin kill switch that takes advantage of extracellular interactions between coral hosts and their microalgae.

Microbe-associated molecular patterns (MAMPs) on Symbiodinium interact with pattern recognition receptors (PRRs) on the corals to facilitate endocytosis and symbiosis. (1) Recent studies have identified mannose recognising Lectin ConA as a conserved MAMP across Symbiodinium sp. (2, 3) Lectin ConA has been implicated in key signalling pathways within other eukaryotes. (4, 5) This makes it an ideal candidate for a potential kill switch within our modified Symbiodinium. Our design includes the induction of CcdB antitoxin production through PRR activated Lectin ConA signalling pathways. (6) This will neutralise CcdA toxin that is constitutively produced under a weak promoter, causing cellular death of the Symbiodinium following detachment from the coral host.

Figure 1: Diagrammatic representation of proposed kill switch. Created with BioRender.

Looking forward, the proposed implementation of our coral bleaching solution would involve the transfer of these genetically modified Symbiodinium into an in situ marine environment. Through consultation with Melissa Katon and Lawrence Menz we have developed a risk-mitigating, three-stage approach. We will monitor the impacts of our modified Symbiodinium on the coral polyp and the surrounding ecosystem at each stage before progressing to the next.

  1. Ex-situ testing of our solution within a controlled and contained environment. This will be conducted at the Sydney Marine Institute in tanks simulating ocean conditions.
  2. In-situ testing in multiple areas of low biodiversity.
  3. In-situ release in an area of ‘normal’ biodiversity.

References

  1. 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.
  2. Logan DDK, LaFlamme AC, Weis VM, Davy SK. 2010. Flow cytometric characterization of the cell-surface glycans of symbiotic dinoflagellates (Symbiodinium spp.). J. Phycol. 46:525–533
  3. 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.
  4. 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.
  5. 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.
  6. 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.

SCIENCE COMMUNICATION

SCIENCE COMMUNICATION

Science communication not only enables collaboration between scientists where research rationales, results and conclusions can be shared, it also bridges the gap in knowledge of a plethora of stakeholders who may not have a science background. Regardless of the ongoing pandemic, our team acknowledged the importance of communicating science to the society. Therefore, we made an attempt to share as much information about synthetic biology and our project to a wide range of audiences from primary school students to the general public.

Based on research, suggestions, and support from the previous UNSW iGEM team members, UNSW Puzzle Society, and UNSW BABSOC (School of Biotechnology and Biomolecular Sciences Student Society), our team created the science communication components. We aimed to gather constructive feedback and spark open discussions about synthetic biology and coral bleaching among target audiences through various modes of communication including written texts, videos, games, and presentations. (1) Thus we created the different components of science communication: children’s book, an education package, a virtual escape room, a recorded interview video with MolecularCloud, and a presentation for the general public. From these, the team hopes that we have and will continue to increase awareness and inspire our community to contribute to the prevention of coral bleaching. Therefore, we also have some potential future collaborations that we have planned with other partners including with the University of Melbourne iGEM team, Student Science Squad, Startup Business International, The Future Project at Kings School, and the Kings School.

Children’s Book

The children’s book was targeted for children of the ages from six to nine. This book aimed to introduce children to 1) The main causes of global warming and 2) The effect it has on coral bleaching. It is both a story book and an activity book which empowers students to suppress global warming and hence coral bleaching.

Education Package

The education package was designed to suit year 11 and 12 students in New South Wales (NSW). It consisted of several components including an instruction page for educators, lecture slides, a supporting document, a physical card game, project brochure, and a feedback form.

Virtual Escape Room

Coral Conundrum is an online escape room which was aimed to expose university students to synthetic biology and our project. It also encouraged attendees to have open discussions about the topics with their teammates as they followed the storyline.

MolecularCloud Interview

An interview session about our project overview was conducted between MolecularCloud and the UNSW 2020 iGEM team. The aim of this interview was to communicate our project about saving corals to the general public.

Presenting to the Public

“How is Science Solving Local and Global Challenges?” was the theme of our presentation and panel discussion about taking on challenges. This was based on our iGEM project and the application of synthetic biology.

References

  1. Borup J, West RE, Thomas R. The impact of text versus video communication on instructor feedback in blended courses. Educational Technology Research and Development. 2015 Apr 1;63(2):161-84.

ACKNOWLEDGEMENTS

ACKNOWLEDGEMENTS AND SPONSORS

Acknowledgements:

Mentors

Dr Dominic Glover, Primary PI
Ahmad Zeeshan Siddiqui, Primary Advisor
Joshua McCluskey, Lab Mentor
Dr Daniel Winter, Lab Mentor
Megan Jones, HP Mentor
Kevin Huynh, HP Mentor
Justine Salazar, Wiki Mentor

Lab Work

A/Prof. David Suggett
Professor Madeline Van Oppen
Associate Professor Wallace Bridge
Melissa Katon
Jesse Bergman
Brian Ee, Molecular Dynamics Advisor
Dr Rodrigo Santibáñez, Dry lab simulation
Professor Mark Tanaka, Mathematical Modelling

Human Practices

Professor Matthew Kearnes
Dr. Aditi Mankad
Elle Davidson
Lawrence Menz
Associate Professor Suhelen Egan
Australian Coastal Local Council
Great Barrier Reef Marine Park Authority (GBRMPA)

Sponsors: