Team:GA State SW Jiaotong/Description
Inspiration For Our Project
Coral reefs are essential to both marine and terrestrial life. They support a quarter of all marine organisms[1], and the impact of these ecosystems extends well beyond the reefs themselves. Over 500 million people rely on corals to provide food and resources[2]. Coral reefs support the global economy, worth $375 billion each year[2], by providing economic goods and services. However, corals have been adversely affected by a global environmental catastrophe known as coral bleaching, where environmental stressors such as excessive seawater temperatures lead to the expulsion of corals’ vital algal symbionts. We first learned about the extent of this problem through a Netflix documentary Chasing Corals, where we learned about the devastating effects of coral bleaching. Although there are current efforts to protect the reefs, such as enforcing marine protected areas that prevent overfishing and polluting industrial activities that harm the corals, these local attempts are still unable to stop the detrimental effects of climate change on ocean waters. After the heatwave in 2016 and 2017, approximately 50% of the Great Barrier Reef bleached[3]. Reef water temperatures around the world are expected to increase even further by 2℃[4]. Without strict proposals to eliminate fossil fuel consumption, marine heatwaves will become more severe[5]. These obstacles, as well as the documentary, inspired our Georgia State University/Southwest Jiaotong University iGEM team to utilize synthetic biology in an attempt to save the corals.
Some symbiotic algae (dinoflagellates) have proven to be more resistant to bleaching than others. However, these algae tend to have slower growth rates. We identified natural heat resistant genes that we plan to transform into fast-growing, bleaching susceptible algae. Then, we will introduce these genetically modified cells back into the corals.
Our plan involved culturing and transforming the microalgae symbiont, Symbiodinium microadriaticum. The first step was to culture the algae. To determine the optimal algal growth conditions, we tested various factors including growth media, temperature, and light intensity. The second step was to transform the algae. We have attempted three different transformation methods: Lonza, Agrobacterium-mediated, and yeast-based electroporation. As a proof of concept, a codon-optimized red fluorescent protein was designed and cloned into a dinoflagellate-optimized expression plasmid (DinoIII)[6] to transform S. microadriaticum using a Lonza nucleofector device, which has been successfully used on another non-symbiotic dinoflagellate (Oxyrrhis Marina). At the same time, our team is attempting to replicate an Agrobacterium tumefacien co-culture transformation done by Ortiz-Matamoros et. al, which utilizes a binary vector, pCB302-GFP-MBD. Their attempt has been the only known successful transformation of Symbiodinium. However, this transformation was unstable since the cells were left unable to divide[7]. The last electroporation is meant for yeast but has been performed on Symbiodinium as well. We used our Dino-RFP to attempt that transformation.
After several unsuccessful attempts to transform Symbiodinium by these various methods, we learned of an issue with the Symbiodinium genome that may be a major obstacle to transformation: Symbiodinium has permanently condensed chromosomes. This is a major problem and probably accounts for the unstable nature of previous transformations because it is nearly impossible to access relevant gene sites that are located within the condensed chromosomes. To overcome this genomic issue, we now proposed to target the chloroplast for transformation rather than the nucleus. Since the chloroplast’s genome is more similar to a prokaryotic genome, it is possible to insert a minicircle of DNA that does not require genomic integration. Researchers from the University of Cambridge successfully and stably (at least 1 year later) introduced a plasmid into the chloroplast of the dinoflagellate A. carterae by utilizing a Biolistics particle delivery system. We initially planned on using their protocol to transform Symbiodinium’s chloroplast but learned that the cost of a store-bought Biolistics gene gun could be upwards of $30,000. Instead, we decided to engineer our own gene gun based on the designs by Worcester’s iGEM team as their design would only cost around $400. We are currently in the process of building this machine and working on improving Worcester's design by increasing stability and power to effectively transform our algae. After we standardize an optimal Symbiodinium transformation using a reporter, we will use this method to introduce bleaching resistance genes into the algae. Our team is working on culturing corals and simulating bleaching events so we can eventually reintroduce our modified algae. The goal is for these corals to take in the newly-engineered algae and express greater resistance to bleaching.