Cloning and Expression of the Iron Regulatory Protein BOL2 From C. glabrata

School Name

South Carolina Governor's School for Science & Mathematics

Grade Level

12th Grade

Presentation Topic

Biochemistry

Presentation Type

Mentored

Abstract

Maintenance of iron levels in the body is crucial as both deficient and excess levels can be detrimental. Having a better understanding of iron signaling pathways helps form the basis for finding treatments for iron-related diseases such as anemia, hemochromatosis, and muscular myopathies. C. glabrata is a haploid, pathogenic organism used as a model system for iron-binding proteins involved in the regulation of iron levels. In C. glabrata, monothiol glutaredoxin Grx4 interacts with the BolA-like protein Bol2 to transfer Fe-S clusters to the nucleus. However, this interaction is not well understood, so it is imperative to study the expression of Bol2 by itself versus the expression of Bol2 with Grx4. From AmplifX software, the expected PCR product size was 297 base pairs. Running a 1% agarose gel confirmed the Bol2 had been amplified through the PCR protocol. Using Double digestion, the two vectors pRSFDuet-1 and pRSFDuet-1-Grx4 were opened to integrate the Bol2 DNA using the restriction enzymes XhoI and PacI. From Gibson assembly, the Bol2 DNA was successfully integrated into the backbones of the two vectors at 50°C. To determine the optimal growth conditions for the new plasmids, pRSFDuet-1-Bol2 and pRSFDuet-1-Grx4-Bol2, they were both transformed to BL21DE3 E. coli cells. Expressions of Grx4 and Bol2 from the pRSFDuet-1-Grx4-Bol2 plasmid and Bol2 from the pRSFDuet-1-Bol2 plasmid were optimized by induction with 0.25 mM IPTG and subsequent growth for 4 hours at 30°C. Future studies will need to characterize the structure of Bol2 and study its interactions with other potential iron-binding proteins.

Location

Furman Hall 118

Start Date

3-28-2020 9:45 AM

Presentation Format

Oral Only

Group Project

No

COinS
 
Mar 28th, 9:45 AM

Cloning and Expression of the Iron Regulatory Protein BOL2 From C. glabrata

Furman Hall 118

Maintenance of iron levels in the body is crucial as both deficient and excess levels can be detrimental. Having a better understanding of iron signaling pathways helps form the basis for finding treatments for iron-related diseases such as anemia, hemochromatosis, and muscular myopathies. C. glabrata is a haploid, pathogenic organism used as a model system for iron-binding proteins involved in the regulation of iron levels. In C. glabrata, monothiol glutaredoxin Grx4 interacts with the BolA-like protein Bol2 to transfer Fe-S clusters to the nucleus. However, this interaction is not well understood, so it is imperative to study the expression of Bol2 by itself versus the expression of Bol2 with Grx4. From AmplifX software, the expected PCR product size was 297 base pairs. Running a 1% agarose gel confirmed the Bol2 had been amplified through the PCR protocol. Using Double digestion, the two vectors pRSFDuet-1 and pRSFDuet-1-Grx4 were opened to integrate the Bol2 DNA using the restriction enzymes XhoI and PacI. From Gibson assembly, the Bol2 DNA was successfully integrated into the backbones of the two vectors at 50°C. To determine the optimal growth conditions for the new plasmids, pRSFDuet-1-Bol2 and pRSFDuet-1-Grx4-Bol2, they were both transformed to BL21DE3 E. coli cells. Expressions of Grx4 and Bol2 from the pRSFDuet-1-Grx4-Bol2 plasmid and Bol2 from the pRSFDuet-1-Bol2 plasmid were optimized by induction with 0.25 mM IPTG and subsequent growth for 4 hours at 30°C. Future studies will need to characterize the structure of Bol2 and study its interactions with other potential iron-binding proteins.