Difference between revisions of "Team:Georgia State/Design"

Our ultimate goal is to save the corals, but how? Where do we start?

We began by first learning about what coral bleaching is and what causes it. After speaking to Alanna Waldman, a marine biology research assistant at Nova Southeastern University, we quickly realized there isn’t just one cause of coral bleaching, which means there isn’t just one way of approaching the problem.

Coral bleaching is when corals are put under some sort of stress such as increased temperatures and acidic ocean waters and, as a result, expel their mutualistic microalgal symbiont, Symbiodinium. This leaves the coral “bleached” and vulnerable to disease. However, coral bleaching is reversible; Once the conditions are favorable again, the coral can reuptake the algae. The main problem facing corals today is that these stressors are present for prolonged periods of time. The longer the corals are without their symbionts, the more likely they are to die by disease or by being inhabited by macroalgae.

Now that we had understood the problem more deeply, we began to brainstorm ways of fixing it. But before we can come up with plans of our own, we have to know more about what is currently being done to address this global phenomenon. So yet again, we spoke to our friend Alanna Waldman, who is involved in these restoration efforts, to learn more. As of right now, there is little research being done on to solve the problem of coral bleaching. There are, however, several ongoing restoration efforts where corals are taken to the lab and their stressor removed to allow their symbiotic algae to be uptaken before they are replanted in-field. However, some issues involved with this is: one, what’s stopping the corals from being bleached again when another stressor reappears, and two, what can speed up restoration efforts to keep up with the rate of coral bleaching worldwide? As of right now, we are simply trying to keep our head above water.

At first we were thinking of simply stopping coral bleaching from happening altogether by reducing the stressors but after speaking with Alanna we revised our initial plan for a more practical one: we will utilize the critical period during a bleaching event for the corals to uptake engineered bleaching-resistant Symbiodinium.

And so we looked into any reports of research involved in the transformations of Symbiodinium and we found two papers, one from 1998 (Ten and Miller) who utilized silicon carbide whiskers, polyethylene glycol (PEG), and vigorous shaking to introduce foreign DNA into Amphidinium sp. and Symbiodinium microadriaticum with a success rate of ~1 ppm. And the other in 2015 (Ortiz-Matamoros et al.) who used glass beads to disrupt the Symbiodinium cells and then a co-incubation with Agrobacterium tumefaciens to transform foreign DNA into Fugacium kawagutii (formerly Symbiodinium kawagutii) and S. microadriaticum. But that is all we could find. We quickly realized our project this year will be more focused on finding an optimal protocol for transforming these microalgae and creating the best methodology and expression plasmid to use.

But before we could transform anything, we would have to first successfully culture these organisms in our lab.

We decided to start by attempting to replicate the latest attempt done by Ortiz-Matamoros et al, however there was no published record of the plasmids they transformed into the Symbiodinium. We reached out to their lab and we got into touch with Dr. Tania Islas at the Instituto de Ciencias del Mar y Limnología, UNAM and she sent us the pCB302-gfp-MBD plasmid they used in their own experiments and advised us on the best way to remove it from the filter paper they sent it to us on. They also sent us the recipe for the ASP-8A media we would need to culture the Symbiodinium in, which we were not able to locate elsewhere.

We planned to test out optimal growth conditions for Symbiodinium, different media, light intensities, and temperature.

Although this was a great starting point, the pCB302-gfp-MBD is not a codon optimized plasmid for dinoflagellates expression but rather optimized for use in plants. An additional issue was the after they performed that transformation, the Symbiodinium was left unable to reproduce and photosynthesize. We hypothesize this could be due to the introduced GFP gene conflicting with cellular processes.

The next step in our project design was to create an optimized dinoflagellate expression plasmid carrying a reporter gene and we found that PhD students Brittany Splecher et. al. at University of Connecticut have made exactly that, the Dino III plasmid carrying a GFP gene which they transformed into a dinoflagellate relative of Symbiodinium microadriaticum, Oxyrrhis marina. We wanted to replace the GFP gene with RFP to see if this would cause the Symbiodinium to be more functional post transformation. So we needed to first remove the GFP part out of the Dino III plasmid and design, synthesize and ligate a codon optimized RFP part in place of it.

To test the functionality of our modified Dino III plasmid, we were going to use the electroporation transformations performed by the University of Connecticut and transform it into Oxyrrhis marina, our model organism which has been successfully and stably transformed and is easy to culture. We plan to send our plasmid to the University of Connecticut lab in hopes they will try to transform our plasmid as well. We planned to test out optimal growth conditions for Oxyrrhis marina as well, different media, light intensities, and temperature.In addition, we will also be culturing Dunaliella tertiolecta, the food source for Oxyrrhis marina.

As we solidify the optimum culturing techniques we plan to create an algae house that will house these algae cultures in flasks in the optimum light intensity and temperature. We also plan to create a predator-prey model for the Oxyrrhis marina and Dunaliella tertiolecta to see how often and how much Dunaliella tertiolecta we should feed the Oxyrrhis marina for optimal growth conditions for Oxyrrhis marina.

Alongside this we planned to test our various electroporation protocols and settings for Symbiodinium in hopes something may work. Then based on the most successful transformation method for Symbiodinium microadriaticum, our project can take one of two routes: either an electroporation is the best transformation method and we will use the Dino III plasmid to introduce genes we believe will help Symbiodinium resist bleaching events or the Agrobacterium mediated transformation is the most efficient transformation method in which case we will modify the pCB302-gfp-MBD plasmid and remove the plant optimized parts and replace them with the dinoflagellate promoter, reporter gene, and terminator found in the Dino III plasmid and see if the reporter gene is expressed. Then once we can prove our new binary plasmid works, we will replace the reporter gene with a gene we believe will aid in coral bleaching resistance.

Then once we’ve got our engineered Symbiodinium, we will introduce it to the coral. But how can we test if the coral will even uptake our algae in the first place? When in doubt, talk to the experts. We reached out to the Coral Husbandry Expert at the Georgia Aquarium, Kim Stone, to learn more about coral. We took a trip to the Georgia Aquarium and talked with Kim Stone and told her about our iGEM project. 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 explained in great detail of what a step by step process would look like to first purposely bleach the coral in a laboratory setting and then slowly allowing the coral to recover and potentially uptake our algae. We even began planning to partner up in the future to set this up when we get to that point in our research efforts.

Another aspect of our project we took into great consideration is safety. Potentially releasing a genetically modified organism outside of the lab should not be handled irresponsibly and safety should be the number one concern. But we weren’t sure what that exactly entails. We looked into other labs that had experience with this and found a lab at the University of San Diego led by Dr. Johnathan Shurin, who were the first to conduct an EPA-approved outdoor experiment using microalgae. We set up a phone interview to discuss how they were able to make their research possible and what measures they had to take to ensure environmental responsibility and safety. They reported back that we would have to get in touch with the Environmental Protection Agency and would have to fill out a “TERA” form for this process to even begin. They also advised that this is a lengthy process. Although this did not affect our project design it did give us more future direction and gave us an approximation of the timeline of our final project.