Functional Analysis of Putative RNase E Protein Isolated from the Bacteriophage ShiVal
Presentation Type
Poster
Faculty Advisor
Quinn Vega
Access Type
Event
Start Date
26-4-2024 2:15 PM
End Date
26-4-2024 3:15 PM
Description
ShiVal is a novel bacteriophage that specifically infects Mycobacterium smegmatis strain MC2 155 to fully complete its replication cycle. Gene 68, which spans the region between base pairs 57,343 and 58,050 in ShiVal’s genome, has been characterized to code for the RNase E protein due to similarities between the base pairs in this gene and those usually conserved in RNase E genes. However, further scientific investigation is needed to make a conclusive statement about its identity. To that end, the gene was ligated into the pGEX vector and Escherichia coli strain BL21 were transformed with the engineered plasmid so that the expression of the RNase E gene product could be induced. Compared to prior attempts, this time the purification of the protein of interest produced a band of higher intensity in a SDS-PAGE protein gel meaning that a higher yield of pure protein was obtained. The purification exploited the affinity chromatography with GSH, the reduced counterpart of the Glutathione S-transferase protein which was fused with the RNase E protein. In-vitro cleavage assays with RNA and RNase E at different temperatures are underway so that more can be learned about the denaturing potential that the protein of interest has.
Functional Analysis of Putative RNase E Protein Isolated from the Bacteriophage ShiVal
ShiVal is a novel bacteriophage that specifically infects Mycobacterium smegmatis strain MC2 155 to fully complete its replication cycle. Gene 68, which spans the region between base pairs 57,343 and 58,050 in ShiVal’s genome, has been characterized to code for the RNase E protein due to similarities between the base pairs in this gene and those usually conserved in RNase E genes. However, further scientific investigation is needed to make a conclusive statement about its identity. To that end, the gene was ligated into the pGEX vector and Escherichia coli strain BL21 were transformed with the engineered plasmid so that the expression of the RNase E gene product could be induced. Compared to prior attempts, this time the purification of the protein of interest produced a band of higher intensity in a SDS-PAGE protein gel meaning that a higher yield of pure protein was obtained. The purification exploited the affinity chromatography with GSH, the reduced counterpart of the Glutathione S-transferase protein which was fused with the RNase E protein. In-vitro cleavage assays with RNA and RNase E at different temperatures are underway so that more can be learned about the denaturing potential that the protein of interest has.