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

So, we looked into any research involved in the transformations of Symbiodinium and found two papers, one from 1998 (Ten and Miller) that 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. The other, published in 2015, (Ortiz-Matamoros et al.) utilized glass beads to disrupt the Symbiodinium cells then co-incubated them with Agrobacterium tumefaciens to transform foreign DNA into Fugacium kawagutii (formerly Symbiodinium kawagutii) and S. microadriaticum. Unfortunately, this was all we could find, so we quickly realized our project would be more focused on finding an optimal protocol for transforming these microalgae and creating the best methodology and expression plasmid to use.

Before we could transform anything, we first had to successfully culture these organisms in our lab. We tested different media, light intensities, and temperature to determine the optimal growth conditions for Symbiodinium. As we solidified the best culturing techniques, we also created an algae house that carried these algal cultures in flasks at the optimum light intensity and temperature.

We then started the transformation efforts by attempting to replicate the latest one done by Ortiz-Matamoros et al; however, there was no published record of the plasmids they transformed into the Symbiodinium. So, we reached out to their lab and got in touch with Dr. Tania Islas at the Instituto de Ciencias del Mar y Limnología, UNAM who 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 that they had given us. They also provided us with the recipe for the ASP-8A media we would need to culture the Symbiodinium in, which we were not able to locate elsewhere.

Although this was a great starting point, the pCB302-gfp-MBD is not a codon-optimized plasmid for dinoflagellate expression but, instead, for plant expression. An additional issue was that after they performed that transformation, the Symbiodinium were left unable to reproduce nor photosynthesize. This may have been 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. 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 replaced the GFP gene with that of RFP to see if this would cause the Symbiodinium to be more functional post-transformation. So, we removed the GFP part out of the Dino III plasmid then designed, synthesized, and ligated a codon-optimized RFP part in place of the GFP one.

To test the functionality of our modified Dino III plasmid, we used the electroporation procedures performed by the University of Connecticut to transform it into Oxyrrhis marina, our model organism, which has been successfully and stably transformed and is easy to culture. We plan on sending our plasmid to the University of Connecticut lab in hopes that they will try to transform our plasmid as well. In addition, we also cultured Dunaliella tertiolecta, the food source for Oxyrrhis marina. We then developed a predator-prey model for the Oxyrrhis marina and Dunaliella tertiolecta to see how often and how much Dunaliella to feed into the system to achieve the highest Oxyrhhis growth rate possible.

Alongside this, we tested out other electroporation protocols and settings for Symbiodinium in hopes that something may work. 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 the Dino III plasmid will be used to introduce genes we believe will help Symbiodinium resist bleaching events, or the Agrobacterium-mediated transformation is the most efficient 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. Once we prove our new binary plasmid works, we would replace the reporter gene with a gene correlated to coral bleaching resistance.

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 corals. We didn’t realize before this talk that getting the coral to uptake the algae would be a grand project in and of 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 devised a step-by-step process beginning with purposely bleaching the coral of its original algae in a closed system then slowly allowing the coral to recover enough to potentially uptake our modified algae. We may partner with the GA Aquarium in the future to do this when we're ready.

Another aspect of our project that 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. We looked into other labs that had experience with this and found one at the University of San Diego led by Dr. Johnathan Shurin, who was 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 taken 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 an approximate timeline of our final project.