Date of Award

2020

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Area of Concentration

Biochemistry

Abstract

Oxygen binding proteins are essential parts of life in aerobic organisms due to their role in carrying oxygen to oxygen deficient parts of the specimen. Globins are a type of oxygen binding protein, known for this behavior as oxygen binders and transporters. Common globin proteins include hemoglobin and myoglobin found in humans. C. elegans is a nematode often used as a model organism as they have similar metabolic pathways in comparison to humans. This nematode was initially assumed to transport oxygen by diffusion due to its small size (1 mm X 80 µm). In this experiment, attempts were made to clone GLB-1, GLB-10, GLB-21, GLB-31 of the 33 globin proteins recently discovered from in silico analysis of the C. elegans genome and to express them in E. coli. RT-PCR was used to amplify the globin genes which were placed into a pET303CT-His plasmid through Gibson Assembly. Transformation of E. coli was performed with successful isolation of the globin proteins, excluding GLB-21. The GLB-1, GLB-10 and GLB-31 proteins were then induced and purified through His-tag purification, yielding 1.17 µg/µL, 1.17 µg/µL and 0.17 µg/µL concentrations determined in a Bradford assay. These 3 proteins were then observed through UV-Visible Spectroscopy, and the results indicated heme binding and oxygen binding. Sodium hydrosulfite was used to obtain the deoxygenated state of the proteins, and over time, the instability of these proteins was observed. Molecular modeling of the globin proteins was observed using homologous proteins, where GLB-1 was a control. Based on amino acid sequence alignments, GLB-10, GLB-21 and GLB-31 did conserve many of the heme binding amino acids observed in other globin proteins.

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