Team:Georgia State/Human Practices

GSU iGEM

Our Experts

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Alanna Waldman

Marine Biologist

Kim Stone

Curator

Dr. Jessica Joyner

Professor

Dr. Jonathan Shurin

Professor

Alanna Waldman

Coral Reef Restoration, Assessment & Monitoring Lab,
Nova Southeastern University

Why we approached her: Alanna is a marine biologist at Nova Southeastern University. Because Alanna is currently working in the field as a research assistant in a Coral Reef Restoration Assessment and Monitoring Lab, we thought she’d be a great person to teach us the basics of coral bleaching

What we asked her about: Why and how coral bleaching occurs, mechanisms of recovery, and the symbiotic relationship between corals and algae.

What we learned: We now understand that the potential causes of coral bleaching remain a mystery, but some likely factors include: extreme temp., high irradiance, prolonged darkness, heavy metal pollution, and pathogenic microorganisms. When the bleaching-inducing stressors disappear, recovery is still not guaranteed. Coral bleaching minimizes the number of fish in the surrounding area. Without fish that consume macroalgae, the already strained corals can be overpowered. On top of this, the gaps between bleaching events are about half as long as they used to be. This isn’t enough time for the corals to fully recover. Alanna also informed us that certain proteins produced by the algal symbionts have been associated with an increased resistance to bleaching. These include: fluorescent proteins, heat shock proteins, and antioxidant enzymes. After learning from Alanna that bleaching is less likely in corals with high algae diversity, we decided to make several different modifications to our symbiodinium rather than just one. We plan on introducing genes that encode fluorescent and heat shock proteins in one alga and antioxidant genes into another. Additionally, we will transform other algae to express a combination of these proteins at varying levels.

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Kim Stone

Curator of Fish and Invertebrates,
Georgia Aquarium

Why we approached her: Kim Stone studies corals at the GA Aquarium, which is only a 20 minute walk from GSU. She’s an expert on coral husbandry and conducts her own sustainability research on these organisms.

What we asked her about: We wanted to learn more about how to care for corals in a lab setting along with how to introduce our modified symbiodinium into them.

What we learned: We had an incredible talk with Kim Stone that ended with a potential partnership in the future. We didn’t realize before this talk that getting the coral to uptake the algae would be a grand project by itself. We thought that we would either dump our modified symbiodinium into a region of coral reef or directly inject the algae into the corals using a syringe-like tool. Stone proposed a much more practical and efficient method. She offered us a system of tanks that can be temperature and light-intensity regulated. We now plan on using these tanks to test a variety of approaches to prompt the coral uptake of our modified algae. We’ll have to start by deliberately bleaching the corals of their existing algae then transfer them to a new tank filled with our transformed symbiodinium. Kim Stone also recommended that we move away from the Aptasia model and shift instead towards a large and stony coral such as Caulastraea furcata. Compared to small soft corals, large stony ones are easier to culture, and early signs of bleaching are not as difficult to detect. Although Aptasia offers these benefits, Caulastraea furcata will be a much better representation of coral species as a whole due to its phylogeny.

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Dr. Jessica Joyner

Professor of Biology,
Georgia State University

Why we approached her: Dr. Joyner is a marine biology professor at our university. We also noticed that she’s an author of a couple publications regarding Agrobacterium Tumefaciens, a bacteria utilized in our project.

What we asked her about: We met with Dr. Joyner expecting to talk about A. Tumefaciens, but she ended up being very knowledgeable about corals themselves.

What we learned: Dr. Joyner introduced the sea anenome Aptasia to us. She suggested we use Aptasia as a model organism to study coral uptake of algae because it is relatively easy to culture and uptakes in a similar way to that of corals. We were initially going to test our modified algae on one of the most common corals in the Carribean: Elkhorn coral, but this organism is much harder to culture and isn’t as easy to obtain because of its position on the critically endangered list. We also learned of the importance of bacterial symbionts that produce mucus layers on the corals to protect from microbial predators.

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Dr. Jonathan Shurin

Vice Chair of Ecology, Behavior & Evolution,
UC San Diego

To inform ourselves about handling genetically engineered algae, we contacted Dr. Jonathan Shurin, the Director of the California Center for algae biotechnology at the University of Southern California (USC). Dr. Shurin and other researchers at USC were the first to conduct an EPA-approved outdoor experiment using microalgae. He and his team worked on culturing several strains of algae in large ponds at USC. Before being able to introduce the modified algae to an external environment, they had to review EPA regulations and fill out a Toxic Substances Control Act (TSCA) Environmental Release Application (TERA) form. The EPA reviews the submitted application and determines if the engineered organism possesses an “unreasonable risk or injury to a human and/or the environment.” He advised our team to plan in advance, as this can be a lengthy process, if we consider introducing our modified algae to another environment. He also provided several tips for culturing the transformed algae including using different types of media (solid or liquid), using constant factors such as temperature and light levels, and to not give up. Considering this was GSU’s iGEM team first time culturing/transforming algae, we were really having a difficult time. He advised us to be patient, and to follow the usual trial and error method of figuring out the most optimal way to grow them. He really emphasized the importance of being able to regulate the growth and dispersion of the modified algae, as it could be a potential risk to other organisms. In their experiment, q-PCR was used to determine when the modified algae had spread into the other bodies of water which they identified as “tank traps.” Preventing contamination was also a big issue to combat when they were growing large cultures, so they were advised by the EPA to rinse material that came into contact with the algae with bleach as well to cover the ponds with bird netting. Speaking to Dr. Shurin really gave us a boost of confidence and led us to really think about the future directions once we successfully transform the algae.

Szyjka, S.J., Mandal, S., Schoepp, N.G., Tyler, B.M., Yohn, C.B., Poon, Y.S., Villareal, S., Burkart, M.D.,Shurin, J.B. and Mayfield, S.P. (2017). Evaluation of phenotype stability and ecological risk of a genetically engineered alga in open pond production. Algal Research, 24, 378–386. doi: 10.1016/j.algal.2017.04.006

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More from Alanna Waldman

Potential causes of coral bleaching:

Extreme temperature

High irradiance

Prolonged darkness

Heavy metals (copper and cadmium)

Pathogenic microorganisms

A lack of algal signaling molecule that would otherwise inactivate coral defenses

Coral reef facts:

Provide $11.9 trillion per year

Support 500 million people worldwide

Over 50% of living coral has been lost

25% of all marine species are at risk

Possible solutions:

Antioxidant enzyme superoxide dismutase

Heat shock proteins

Stress proteins hsp60 and hsp70

Fluorescent proteins

Mycosporine-like amino acids...natural sunscreens

More lipids or low molecular weight lipophilic compounds

More membrane stability = less ROS

Damage to the algal photosystem II (PSII) may be caused by an excess of reactive oxygen species (ROS) produced by the zooxanthellae.

The interval between bleaching events is half as long as it was before. This is not enough time for the corals to fully recover.

Overgrowth of macroalgae may prolong the time it takes for the corals to fully recover.

Ribotype B corals tend to resist bleaching more than ribotype C corals

Biodiversity of corals within a reef enhances their growth and the survival of their tissues while preventing an overgrowth of macroalgae

A coral polyculture is more likely to survive than a monoculture

Repetitive but non-fatal bleaching events may increase the likelihood that a coral with resist heat stress in the future, but not if this is due to increased sedimentation.

Corals grow the fastest between 25-27°C then slow down once 28° C is reached.

Coral larvae are produced in a variety of different sizes and symbiont densities, and their survival is dependent on the specific environment they’re in.

Coral larvae with high symbiont densities were 5x less likely to resist heat stress compared to those of low densities.

More from Kim Stone

Kim Stone is a curator of fish and invertebrates at the GA Aquarium, which is less than a 20 minute walk from GSU. We had an incredible talk with her, and she walked us through some of the basics of coral husbandry. We may even partner with the GA aquarium in the future when we’re ready to test our modified algae in a closed coral reef system. From Kim Stone, we learned: No one knows if coral expel their algae or if the algae themselves leave the symbiotic relationship Factors regarding corals uptaking algae: Energy expenditure Is the energy wasted on uptaking algae worth it if the coral is already in a healthy symbiotic relationship? So, if the coral is healthy with our modified algae, the algae will most likely not be expelled for other, natural algae. The mother coral’s algal symbiont The mother passes on her symbiont to her offspring The offspring can also uptake algae from the environment. Coral Husbandry Protein skimmers are devices that remove organic compounds from the water. It balances nutrients, pH, and organic materials in the tank. A small number of storms can be good for corals because it changes the water, which can eliminate sedimentation/pollution buildup. Also, this allows for asexual fragmentation to occur, which can disperse corals around an environment. Fluorescence can be used as a measure of health High fluorescence= good health Coral Model Organism Large polyp indo-specific stony coral would work best. For example, Caulastrea. Hard/stony because it’s easy to see any signs of bleaching before the coral actually dies. A small coral is hard to observe and culture How to induce the coral to uptake our modified algae: Stress the corals enough to bleach them but not kill them. Do this by increasing the heat of system gradually by 2°F every other day until signs of bleaching (at about 84-86°F) Hold the temperature at 85°F for a couple of days unless signs of bleaching occur sooner In another tank, maintain the temperature at 85°F . Place the, now bleached, coral in. Gradually bring the temperature down to optimal levels. Add algae when the temperature reaches the algae’s optimal level. Have multiple tanks with various algal densities to determine the best concentration Monitor the attempted-modified coral polyps If there are new polyps, Verify if our algae are there. No one knows if this will work. Notes: Use 10-gallon tanks Certain artificial lights can harm the corals The tanks need water flow. If not, one must change the water every day Monitor pH, NH3, etc. using water test kits commonly found at fish stores Target feeding can decrease the amount of maintenance required. Aeration is essential. To eliminate any extra variables, use the same coral species and number/size of the colonies. Integrated Human Practices Detailed: Model Organism Initially, we were going to introduce our algae to one of the most common coral species in the Carribean: Elkhorn coral. After speaking with Dr. Joyner at GSU, she suggested that we use the sea anemone Aptasia as our model organism instead. Aptasia is easy to culture and uptakes algae during both its adult and larval life forms just like coral do. However, Kim Stone, a coral specialist, recommended Caulastraea furcata, which is a coral commonly found in the Indo-Pacific region. Corals found in this area are typically easier to obtain. Caulastraea is also a large stony coral. It’s easier to see early signs of bleaching in hard/stony corals compared to softer ones. Additionally, smaller corals are harder to observe and culture than larger ones are. Algae Diversity At the beginning of this project, we had a plan to introduce a single modification into a single algal species to incorporate into the coral-algae symbiosis. However, we learned from Alanna Waldman that a broader range of algal diversity typically leads to healthier, more bleaching-resistant corals. So, we changed our plan to include a variety of modifications into several different Symbiodinium. For example, we may introduce genes that encode for greater production of fluorescent proteins into one alga while we introduce genes for more heat shock proteins into another alga. We may even go further by having a spectrum of protein production in each alga. So, one alga may produce a few fluorescent proteins and a multitude of heat shock proteins while another could produce a large number of fluorescent proteins and a small number of heat shock proteins. Water Flow We previously thought that corals were very gentle animals that require as little water disturbance as possible. The original plan was to place a coral into a tank with stagnant water. However, Kim Stone informed us that this isn’t the best method. Corals actually need constant water flow to circulate nutrients and prevent sedimentation. In fact, a small number of storms can actually benefit the corals by clearing out some of the sedimentation/pollution and encourage asexual fragmentation to occur. So, we will now place our model organism into a tank with constant water flow. Algae uptake Once our algae have been successfully engineered to resist the stressors that cause bleaching, we will need the coral to uptake them. We thought of several ways of doing this. One way involved dumping a large concentration of our algae into a sea region where a large but stressed coral reef is found. Another possibility was to inject the algae into the coral with a syringe-like tool. After speaking with Kim Stone at the GA Aquarium, we came up with a much more practical solution. We may partner with the GA Aquarium in the future when they can provide us with tank systems that can be regulated with respect to temperature, light intensity, and water flow. We plan on setting up two tanks of seawater. We’ll begin by placing a Caulastraea coral into the first tank. After inducing bleaching by gradually bringing the temperature up 2°F every other day until about 86 °F is reached, we will transfer the organism into a fresh tank. To prevent the coral from going into shock, this fresh tank will start at 86 °F, and its temperature will eventually be brought down to about 72 °F. We will then introduce our algae of varying concentrations and monitor the coral for uptake. If the coral uptakes, we will later observe its offspring and note if the modified symbionts were passed down from the mother.

More from Dr. Jessica Joyner

Dr. Joyner is a marine biology professor at our school, GA State University. After learning about the general mechanisms of bleaching from Alanna, we asked Dr. Joyner more specific questions about our project. For example, what factors contribute to the uptake of algae? Additionally, what model organisms would work best for testing the uptake of our modified algae by the coral? From Dr. Joyner, we learned: Coral bacterial symbionts that produce the mucus layer on coral protect from predation by other microbes Corals are selective when uptaking algae Clade by clade variations Aptasia is a good model organism for uptaking algae because both the larval and adult forms of this organism are easy to culture in the lab.