College of Science and Engineering Projects and Theses
Permanent URI for this collectionhttps://hdl.handle.net/10657.1/830
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Browsing College of Science and Engineering Projects and Theses by Author "Ariza, Martha"
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Item Functional Characterization of Pol-dUT Fusion Enzymes from Pyrococcus Furiosus(2018-05-10) Dash, Asim Kanti; Rashid, Bazlur; Wang, Daniel; Ariza, MarthaAlthough polymerase chain reaction is a powerful technique, often polymerizing errors can jeopardize the whole experiment especially when to study a cloned gene product. Long PCR with conventional Taq DNA polymerases is one such example, where conversion of dCTP to dUTP can increase the error frequency and dUTP incorporation as well. However, using proofreading polymerases for long PCR, the polymerization rate slows down significantly due to incorporation of dUTP during synthesis followed proofreading. To alleviate this problem, now a day we use dUTPase enzyme in the PCR. To have similar effect, our laboratory has designed two fusion enzyme constructs of both a proofreading polymerase and a dUTPase from Pyrococcus furiosus. We are reporting here that both these fusion constructs could remove dUTP from PCR reactions and able to amplify. This project particularly entails successful cloning, expression and purification of a dUTPase enzyme followed by its functional characterization in parallel with the above mentioned fusion enzymes.Item Identification of culturable bacteria isolated from sponges affected in the 2016 massive die-off at the Flower Garden Banks National Marine Sanctuary (FGBNMS)(2017-12-06) Pandya, Dhatri Amitkumar; Santiago-Vázquez, Lory; Ariza, Martha; LaMontagne, MichaelThis project was conducted to identify culturable bacteria isolated from sponges affected in the 2016 massive die-off at the Flower Garden Banks National Marine Sanctuary (FGBNMS). The FGBNMS was considered a healthy reef system until sport divers reported on July 25th 2016 green, hazy water with huge patches of white mats on corals, sponges, other vertebrates and dead animals littering the bottom of East Flower Garden Bank. To help elucidate the root cause of this event, affected and unaffected sponge samples of Agelas clathrodes and Xestospongia muta sponges from East bank were examined for their associated microbial communities using culture-dependent methods. MALDI – TOF – MS and 16S rDNA sequencing were used to identify representative isolates. MALDI – TOF – MS and 16S rDNA sequencing analysis showed the presence of Bacillus firmus, Pseudovibrio spp., Halanaerobium spp., Microbulbifer variabilis, and Microbulbifer spp. in unaffected samples. Bacillus firmus could help sponges by nitrogen fixation and producing secondary metabolites that protects sponges from predators and Halanaerobium sehlinense is fermentative bacteria. The role of Halanaerobium sehlinense in sponges is not yet clear. Affected samples showed the presence of Vibrio spp. and Halanaerobium sehlinense. Vibrio spp. which occurs naturally in sea water as opportunistic pathogenic bacteria exhibits stronger proteolytic (caseinase), phospholipase and hemolytic activities. These potential pathogens may have contributed into massive die-off at East bank of FGBNMS.Item Recovery of Xestospongia muta and Agelas clathrodes Bacterial Microbiome Since the 2016 Mortality Event at the Flower Garden Banks National Marine Sanctuary(2020-05-28) Peterson, Charis Ann; Santiago-Vázquez, Lory; Ariza, Martha; LaMontagne, MichaelOn July 25, 2016, divers discovered a large localized mortality event at the East Bank of the Flower Garden Banks National Marine Sanctuary. This event killed 80% of invertebrates on the site. This sanctuary is a complex ecosystem where sponges play an essential role as members of the reef. The project analyzes the state of recovery of the bacterial microbiome of the sponges Xestospongia muta and Agelas clathrodes, since the 2016 mortality event. During the Fall of 2018, divers collected samples of these sponge species from the East Bank, location of 2016 mortality event, and from the West Bank, unaffected during the mortality. A culture dependent approach was used to determine the microbiome of the sponges. The 16S rRNA gene of the cultures was sequenced to identify the bacteria. Alpha-Proteobacteria and Gamma-Proteobacteria were the most common class of bacteria found in X. muta and A. clathrodes in both the East Bank and the West Bank. A comparison of the 2018 samples to the communities of the 2016 affected East Bank and unaffected East and West Bank will allow us to determine a potential recovery at the level of bacterial symbionts. Since the microbiome data presented only covers years 2016 and 2018, More research in microbial analysis is needed to determine the overall status of the FGBNMS reef and to establish a baseline for normal conditions.