Molecular characterization of the Terminase enzyme from the Shival phage

Presentation Type

Abstract

Faculty Advisor

Quinn Vega

Access Type

Event

Start Date

25-4-2025 12:00 PM

End Date

25-4-2025 1:00 PM

Description

Over the past few decades, antibiotic-resistant bacteria have been evolving at alarming rates. However, research shows that bacteriophages are a promising alternative in treating bacterial infections. To implement phages as permanent forms of treatment, further research is required to understand the mechanisms behind their development and function. At Montclair State University, several phages, specifically the Shival phage, have been isolated and their genomes sequenced. Shival has 101 predicted genes, including gene 2 which is predicted to code for the protein terminase. Currently, all protein predictions have been determined by sequence comparison rather than measuring protein function. Terminase is believed to pack the viral genome into the capsid of the phage. To analyze terminase’s function, the gene was amplified through PCR and inserted into a GST-tag expression vector. The ligated product was then transformed into DH5alpha cells and the plasmids from the resulting transformants were analyzed through restriction digestion. The plasmid was then transformed into BL21 cells for protein. Thus far, the gene has been cloned and partially sequenced, confirming the correct gene was isolated. Although the protein of the correct size has been produced, the protein appears to be insoluble. In response, the common domain that has been aligned with other terminases will be isolated and expressed to increase solubility and aid in purification. Once the protein is purified, protein function analysis will begin by measuring ATPase activity. Through our research, we hope to further our understanding regarding bacteriophage and contribute to its use in medical treatments within the future.

Comments

Poster presentation at the 2025 Student Research Symposium.

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Apr 25th, 12:00 PM Apr 25th, 1:00 PM

Molecular characterization of the Terminase enzyme from the Shival phage

Over the past few decades, antibiotic-resistant bacteria have been evolving at alarming rates. However, research shows that bacteriophages are a promising alternative in treating bacterial infections. To implement phages as permanent forms of treatment, further research is required to understand the mechanisms behind their development and function. At Montclair State University, several phages, specifically the Shival phage, have been isolated and their genomes sequenced. Shival has 101 predicted genes, including gene 2 which is predicted to code for the protein terminase. Currently, all protein predictions have been determined by sequence comparison rather than measuring protein function. Terminase is believed to pack the viral genome into the capsid of the phage. To analyze terminase’s function, the gene was amplified through PCR and inserted into a GST-tag expression vector. The ligated product was then transformed into DH5alpha cells and the plasmids from the resulting transformants were analyzed through restriction digestion. The plasmid was then transformed into BL21 cells for protein. Thus far, the gene has been cloned and partially sequenced, confirming the correct gene was isolated. Although the protein of the correct size has been produced, the protein appears to be insoluble. In response, the common domain that has been aligned with other terminases will be isolated and expressed to increase solubility and aid in purification. Once the protein is purified, protein function analysis will begin by measuring ATPase activity. Through our research, we hope to further our understanding regarding bacteriophage and contribute to its use in medical treatments within the future.