Western Kentucky University

Functional Analysis of a Bacteriophage Gene Product with Cytotoxic Effects on Mycobacterium smegmatis

Grade Level at Time of Presentation

Sophomore

Major

Molecular Biology

Institution 24-25

Western Kentucky University

KY House District #

5

KY Senate District #

27

Department

Dept. of Biology

Abstract

Antibiotic resistance is a critical global health challenge, with the UN estimating that drug-resistant infection deaths will surpass cancer deaths by 2050. Bacteriophages offer a potential alternative to traditional antibiotics, which are becoming increasingly ineffective due to resistance. MooMoo is a mycobacteriophage that is genetically distinct from all known phages, exhibiting <35% DNA similarity to phage genomes in Genbank. Bioinformatic analysis identified several MooMoo genes, including MooMoo gene 51 (MM51), as having no known function. MM51 was later shown to be extremely toxic to M. smegmatis. Using a bacterial two-hybrid assay, this project aimed to characterize MM51 by identifying interactions between the phage and host proteins, revealing mechanisms of MM51’s toxicity. Identifying the host-interacting partner could lead to the development of a new therapeutic. The MM51 gene sequence was previously cloned into the pExTra shuttle vector via isothermal assembly. After confirming the protein's toxicity to host cells, the gene was subcloned into the p2Hα two-hybrid vector and transformed into chemically competent E. coli cells. After clone verification, the two-hybrid assay was performed. A positive interaction was detected by blue colony color in the presence of the antibiotic carbenicillin. Clones that produced a positive interaction were sequenced to identify the host gene. Six out of sixteen clones produced positive interactions, but each clone contained a different host gene fragment. These results suggest that MM51 has multiple interacting partners.

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Functional Analysis of a Bacteriophage Gene Product with Cytotoxic Effects on Mycobacterium smegmatis

Antibiotic resistance is a critical global health challenge, with the UN estimating that drug-resistant infection deaths will surpass cancer deaths by 2050. Bacteriophages offer a potential alternative to traditional antibiotics, which are becoming increasingly ineffective due to resistance. MooMoo is a mycobacteriophage that is genetically distinct from all known phages, exhibiting <35% DNA similarity to phage genomes in Genbank. Bioinformatic analysis identified several MooMoo genes, including MooMoo gene 51 (MM51), as having no known function. MM51 was later shown to be extremely toxic to M. smegmatis. Using a bacterial two-hybrid assay, this project aimed to characterize MM51 by identifying interactions between the phage and host proteins, revealing mechanisms of MM51’s toxicity. Identifying the host-interacting partner could lead to the development of a new therapeutic. The MM51 gene sequence was previously cloned into the pExTra shuttle vector via isothermal assembly. After confirming the protein's toxicity to host cells, the gene was subcloned into the p2Hα two-hybrid vector and transformed into chemically competent E. coli cells. After clone verification, the two-hybrid assay was performed. A positive interaction was detected by blue colony color in the presence of the antibiotic carbenicillin. Clones that produced a positive interaction were sequenced to identify the host gene. Six out of sixteen clones produced positive interactions, but each clone contained a different host gene fragment. These results suggest that MM51 has multiple interacting partners.