Expression of Fra2-Grx4 for Interaction Studies of Fep1 Mediated Iron Homeostasis in S. pombe
School Name
South Carolina Governor's School for Science and Mathematics
Grade Level
12th Grade
Presentation Topic
Biochemistry
Presentation Type
Mentored
Abstract
Iron maintenance is needed for the survival of S. pombe, a nonpathogenic yeast. Iron buildup can be toxic to the cell, while iron shortage can impair essential iron-dependent metabolic pathways. S. pombe has similar iron homeostasis pathways to pathogenic yeasts, and studying S. pombe can help determine the mechanisms governing these pathways. Transcription factor Fep1 is a repressor of iron uptake genes and reduces iron intake during high Fe conditions. This research focuses on the expression of Fep1 and Fra2-Grx4 to study specific protein-protein interactions involved. To do this, we first needed to remove the His-tag, a chain of six histidines, from Fra2. We created a Fra2-Grx4 insert through a gradient PCR reaction. Then we moved on to making the DNA backbone through a restriction enzyme digestion. After successfully producing the two DNA fragments, we combined them through a HiFi assembly. When the plasmid was created, we checked the plasmid with another restriction enzyme digestion. Confirmed samples were checked for point mutations that may keep the proteins from forming. Finally, we performed expression trials to find the best conditions to produce the proteins in high levels. We found Fep1-Fra2-Grx4 is best coexpressed at 30℃ at 150rpm with 1 mM IPTG. This allows further studies between the proteins to determine if they work in complex and what their specific interactions are. Studying the interactions between these proteins can shed light on pathogenic yeasts and ways to combat them in cases of infection.
Recommended Citation
Mbu, Tiku, "Expression of Fra2-Grx4 for Interaction Studies of Fep1 Mediated Iron Homeostasis in S. pombe" (2026). South Carolina Junior Academy of Science. 32.
https://scholarexchange.furman.edu/scjas/2026/all/32
Location
Furman Hall 107
Start Date
3-28-2026 10:00 AM
Presentation Format
Oral Only
Group Project
No
Expression of Fra2-Grx4 for Interaction Studies of Fep1 Mediated Iron Homeostasis in S. pombe
Furman Hall 107
Iron maintenance is needed for the survival of S. pombe, a nonpathogenic yeast. Iron buildup can be toxic to the cell, while iron shortage can impair essential iron-dependent metabolic pathways. S. pombe has similar iron homeostasis pathways to pathogenic yeasts, and studying S. pombe can help determine the mechanisms governing these pathways. Transcription factor Fep1 is a repressor of iron uptake genes and reduces iron intake during high Fe conditions. This research focuses on the expression of Fep1 and Fra2-Grx4 to study specific protein-protein interactions involved. To do this, we first needed to remove the His-tag, a chain of six histidines, from Fra2. We created a Fra2-Grx4 insert through a gradient PCR reaction. Then we moved on to making the DNA backbone through a restriction enzyme digestion. After successfully producing the two DNA fragments, we combined them through a HiFi assembly. When the plasmid was created, we checked the plasmid with another restriction enzyme digestion. Confirmed samples were checked for point mutations that may keep the proteins from forming. Finally, we performed expression trials to find the best conditions to produce the proteins in high levels. We found Fep1-Fra2-Grx4 is best coexpressed at 30℃ at 150rpm with 1 mM IPTG. This allows further studies between the proteins to determine if they work in complex and what their specific interactions are. Studying the interactions between these proteins can shed light on pathogenic yeasts and ways to combat them in cases of infection.
