Although this year we were unable to get into the lab as often as we would have liked, we focused on the research phase of our project. One aspect of modifying the symbiotic algae is performing a genomic analysis on different Symbiodinium clades in order to identify bleaching resistant genes that we can insert or regulate into the algal genome. During our research, we learned about the transcriptome of Symbiodinium, discovered drafts of its genome, and performed a phylogenetic analysis of stress and metabolic genes in Symbiodinium. This led us to hypothesize that the regulation of heat shock proteins and heat shock factors play a role in thermal stress of the algae.
In addition to identifying bleaching resistant genes in the algal genome, we designed experiments to successfully transform Symbiodinium. We engineered a codon optimized RFP reporter gene and inserted it into a dinoflagellate-optimized expression plasmid called DinoIII in order to confirm if our transformation was successful using flow cytometry. With this plasmid, we have attempted to transform the algae by electroporation via Lonza-4D Nucleofector, agrobacterium mediation, glass beads mediation, silica carbide whiskers, and yeast electroporation via Bio-Rad.
From these transformations, we learned the plasmid we designed, DinoIII RFP cannot be used to yield accurate flow cytometry results because Symbiodinium naturally expresses red autofluorescence. Subsequently, we improved our plasmid by engineering a GUS reporter gene into DinoIII, which will produce a distinguishable blue pigment when measured via GUS assay. Also, we have learned that the transformation methods performed on Symbiodinium may not be feasible for delivering genes to its nucleus. Additional research revealed that Symbiodium has permanently condensed nuclear chromosomes, so there is limited access to relevant gene sites for the transient genes to be integrated into the genome. Instead, we want to target the DNA containing chloroplasts of Symbiodinium using a biolistics gene gun particle delivery system. Using this method, we will be able to insert our plasmid without integrating it into the nuclear genome.