Extrachromosomal circular DNA (eccDNA) has been identified in a broad range of eukaryotes and have been shown to carry genes and regulatory sequences. Additionally, they can amplify within a cell by autonomous replication or reintegration into the genome, effectively influencing copy number in cells. This has significant implications for cancer, where oncogenes are frequently amplified on eccDNA. However, little is known about the exact molecular mechanisms governing eccDNA functionality. To this end, we constructed a fluorescent reporter at an eccDNA-prone locus of the yeast genome, CUP1. It is our hope that this reporter will contribute to a better understanding of eccDNA formation and amplification within a cell.

Oxymonas is a genus of Oxymonad protist found in the hindgut of drywood termites (family Kalotermitidae). Many genera of drywood termites are invasive pests globally. The hindgut microbiome of Cryptotermes brevis, the West Indian drywood termite, has not been described in detail, and only one published sequence exists of Oxymonas from C. brevis. This study aims to analyze Oxymonas sequences in C. brevis from whole gut genetic material, as well as to dissect its place in phylogenetic trees of Oxymonas and how it fits into specific and evolutionary patterns. To amplify the 18S rRNA gene Oxymonas from C. brevis, the MasterPure DNA extraction kit was used, followed by PCR amplification, followed by agarose gel electrophoresis, followed by purification of the resulting gel bands, followed by ligation/transformation on to an LB agar plate, followed by cloning the resulting bacterial colonies, and topped off by colony screening. The colony screening PCR products were then sequenced in the Genomics Core, assembled in Geneious, aligned and trimmed into a phylogenetic tree, along with several long-read amplicon sequences from Oxymonas in other drywood termites. All whole gut sequences and one amplicon from C. brevis formed a single clade, sharing an ancestor with a sister clade of Oxymonas sp. from C. cavifrons and Procryptotermes leewardensis, but the other long-read fell into its own clade in a different spot on the tree. It can be conjectured that the latter sequence was contaminated and that the C. brevis clones are a monophyletic group, a notion further corroborated by a distantly related clade featuring sequences from Cryptotermes dudleyi, which in turn has a sister taxon of Oxymonas clones from C. cavifrons and P. leewardensis, pointing toward a different kind of co-diversification of the hosts and symbionts rather than cospeciation.

The objective of this study is to create a spectrophotometric assay that can measure quinone reduction in the HbRC. The key techniques used in the project consisted of a PCR, a pseudo golden gate, a transformation into E. coli, a conjugation into Heliomicrobium modesticaldum, a growth study, a HbRC prep, and absorbance spectroscopy. PCR was crucial for amplifying the Cyt c553-PshX gene for the pseudo golden gate. The pseudo golden gate ligated Cyt c553-PshX into the plasmid pMTL86251 in order to transform the plasmid with the desired gene into the E. coli strain S17-1. This E. coli strain allows for conjugation into H. modesticaldum. H. modesticaldum cannot uptake DNA by itself, so the E. coli creates a pilus to transfer the desired plasmid to H. modesticaldum. The growth study was crucial for determining if H. modesitcaldum could be induced using xylose without killing the cells or inhibiting the growth in such a way that the project could not be continued. The HbRC prep was used to isolate and purify the Cyt c553-PshX protein. Absorbance spectroscopy and JTS kinetic assay was used to characterize and confirm that the protein eluted from the affinity column was Cyt c553-PshX. The results of the absorbance spectra and JTS kinetic assay confirmed that Cyt c553-PshX was not made. The study is currently being continued using a new system that utilizes SpyCatcher SpyTag covalent linkages in order to attach cytochrome to reduce P800 to the HbRC.