Team:GA State SW Jiaotong/Improvement
Improvement of an Existing Part
Our iGem project in the previous years focused on modifying symbiotic algae to tackle the issue surrounding coral bleaching. We hypothesized that we could regulate thermal stress in algae using the heat-resistant genes identified in the algal genome and transformed it into our dinoflagellate, Symbiodinium. However, the DinoIII RFP plasmid that we designed expresses a red fluorescence, and we later found out that Symbiodinium also naturally expresses red autofluorescence. In addition, the DinoIII GFP plasmid we synthesized in the past year wasn’t able to help identify whether the final transformation into Symbiodinium was successful due to the interfering of the natural background color of the algae under microscope. Given the results we gathered in the previous iGem competitions, we suggested engineering a GUS(B-glucuronidase) reporter gene into the DinoIII plasmid, which expresses a visible blue pigment in a GUS assay. The original GUS sequence chosen were from the PBI121 GUS plasmid. Over the summer, our team successfully inserted the GUS gene into the DinoIII plasmid, transformed into E.coli and Agrobacterium Tumefaciens, although transformation into Symbiodinium was still an issue.
In our project, the Dino-III plasmid was kept in the design to aid in delivery of the target gene, and ultimately, expression of B-glucuronidase. This specific plasmid was originally chosen because there was enough previous research to build upon. In addition, it has the promoter that theoretically can help with the B-glucuronidase expression in the aglae. Moreover, it contains a segment of the DNA sequence similar to a portion in the Symbiodinium DNA sequence which theoretically will help with the horizontal gene transfer into an eukaryotic organism through transformation. It is the only dinoflagellate-optimized plasmid for which publications exist to support its use for transformation. The framework was also designed to be used as a general plasmid for the transformation of many dinoflagellate species by researchers from the University of Connecticut.
Codon Optimization
Codon optimization was also used to further improve the constructed DinoIII-GUS plasmid. This was done in the hopes of increasing efficiency of translation and further to improve the expression of this gene in the transformed organism. The DinoIII-GUS plasmid was, therefore, codon optimized for transformation into the well defined species Symbiodinium kawagutii. Snapgene was further used as the tool to constantly check the updated codon frequency after each nucleotide modification.
