Travel Report: Cassandra Carroll, Biology
Cassandra Carroll, a Doctoral student in Biology, received a Graduate International Research Travel Award (GIRTA) to further her research in Scotland, UK. Her report:
From January to April 2015, I was as the University of St. Andrews in Scotland, UK conducting research for my PhD project. Part of my work involves characterizing the enzyme involved in siderophore biosynthesis in the pathogenic fungus, Rhizopus delemar. Siderophores are small molecules that chelate iron and supply it to the microbe; consequently, production of siderophores is part of the disease-causing process of many microorganisms, including fungi. By characterizing the structure and function of the siderophore biosynthetic enzyme in R. delemar, I will facilitate the development of enzyme inhibitors. Such inhibitors may reduce the morbidity and mortality of fungal infections due to R. delemar.
The Biomedical Sciences Research Complex (BRSC) at the University of St. Andrews specializes in X-ray crystallography for the purpose of probing biological mechanisms and drugtarget pathways. During my studies at the BSRC, I was able to complete four of my five original aims:
- Successfully over-express all Rfs proteins (wild type and mutant enzymes) in E. coli
- Purify Rfs proteins to sufficient quantity for crystallization trials
- Confirm activity of Rfs proteins
- Use bioinformatics and protein modelling software to determine the structure of Rfs and mutants
- Crystallize Rfs and mutant proteins
I determined the optimal conditions for overexpression and purification of my proteins in E. coli. Final concentrations for Rfs and mutant proteins ranged from 15-35 mg/ml. The purified proteins were active, with the exception of one mutant; however, this mutant was predicted to be nonfunctional as it contains a mutation in the enzyme active site. I set up all purified proteins in crystallization trials using commercially-available screens available in the BRSC. In total, 384 different crystallization conditions were tested for each protein. Crystallization of Rfs and mutant proteins occurred; however, they were not suitable for doing X-ray crystallography. The project is being continued in collaboration with the Naismith lab at the University of St. Andrews.
During my time at the University of St. Andrews, I learned many essential skills necessary to complete my PhD research. I was able to expand my knowledge on protein purification (e.g., protein induction, nickel pull down assays, and column chromatography). I was also introduced to many new techniques (e.g., circular dichroism spectroscopy, gel filtration, liquid chromatography-mass spectrometry and the use of crystal imaging software). Not only did my time in Scotland improve my skill set as a researcher, it established a research support network and has allowed for continued collaboration with scientists at the BSRC. I believe this will greatly enhance the impact of my research.
Along with the research skills that I gained, travelling to Scotland also provided me with great personal growth. Establishing myself in a new country and becoming familiar with their culture and history was a very rewarding experience.
I am grateful to SFU for the financial support that allowed me to undertake this research. I would like to express my appreciation to Drs. Jim Naismith and Huanting Liu at the BSRC for their expert guidance and use of their resources, as well as the rest of the BSRC research team for advice and assistance both in and out of the laboratory. Finally, I would like to thank Dr. Margo Moore for providing me with this opportunity and for supporting me throughout my PhD career.
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