Genome Annotation of Bacteriophages Allegro and Moostard

During the spring semester of the one-year-long HHMI SEA-PHAGES experience, we annotated the sequenced genomes of two novel Mycobacterium smegmatis bacteriophages, Allegro and Moostard. Allegro is a member of the Mycobacterium Cluster B and a member of the sub-cluster B2. Allegro’s genome is 67,439 bp in length and has approximately 92 predicted genes. Moostard is a member of Mycobacterium Cluster L and a member of the sub-cluster L3. Its genome is 69,480 bp in length and has approximately 108 predicted genes. Using DNA Master, Phamerator, and Starterator, along with homology comparisons to databases at HHPred and NCBI, the putative open reading frames were assessed for designated location and function. Multiple students called each gene allowing for an independent assessment of the reading frames. The data from all students were merged and assessed. Moostard encoded for integrase proteins which suggest a temperate lifestyle. Allegro did not contain an integrase protein but did encode for a putative immunity suppressor protein. The comparative analysis of phage genomes helps expand our understanding of the genomic diversity of bacteriophages found in this region of South Carolina and successfully introduces a genuine research experience to students in their first year of undergraduate studies at Winthrop University

Genome Annotation of Bacteriophages Allegro and Moostard

During the spring semester of the one-year-long HHMI SEA-PHAGES experience, we annotated the sequenced genomes of two novel Mycobacterium smegmatis bacteriophages, Allegro and Moostard. Allegro is a member of the Mycobacterium Cluster B and a member of the sub-cluster B2. Allegro’s genome is 67,439 bp in length and has approximately 92 predicted genes. Moostard is a member of Mycobacterium Cluster L and a member of the sub-cluster L3. Its genome is 69,480 bp in length and has approximately 108 predicted genes. Using DNA Master, Phamerator, and Starterator, along with homology comparisons to databases at HHPred and NCBI, the putative open reading frames were assessed for designated location and function. Multiple students called each gene allowing for an independent assessment of the reading frames. The data from all students were merged and assessed. Moostard encoded for integrase proteins which suggest a temperate lifestyle. Allegro did not contain an integrase protein but did encode for a putative immunity suppressor protein. The comparative analysis of phage genomes helps expand our understanding of the genomic diversity of bacteriophages found in this region of South Carolina and successfully introduces a genuine research experience to students in their first year of undergraduate studies at Winthrop University

Characterizing the Effect of Bacteriophage Cain Genes on Mycobacterium smegmatis

Bacteriophages are viruses that use bacteria to replicate and are studied as a model for host-pathogen interactions and co-evolution. The HHMI SEA-GENES program aims to contribute to this body of knowledge by evaluating phage gene function. This particular study intends to determine gene functions of phage Cain, a virus that targets Mycobacterium smegmatis , through molecular cloning and cytotoxicity assays. Molecular cloning, the combination of pExTra plasmid and target genes, creates a product that can then be used in these phenotypic assays. Genes are individually expressed in M. smegmatis to identify those that modify bacterial growth and fitness. To initiate the cloning process, individual Cain genes were amplified using polymerase chain reaction, then verified via gel electrophoresis. The samples were then purified for isothermal assembly, which is the construction of a plasmid from purified DNA and plasmid backbone. The assembled plasmid was integrated into E. coli and M. smegmatis colonies via bacterial transformation. Through clone verification, colonies carrying the gene-specific plasmid were tested to confirm they contained the inserted gene. Research progress was measured by several benchmarks, including successful amplification of Cain genes, assembly of genes in the pExTra plasmid, E. coli and M. smegmatis transformation, and completion of a cytotoxicity assay. Future directions of this research include additional phenotypic assays that investigate phage defense mechanisms, and studies that asses the physical interaction between Cain and M. Smegmatis proteins

Isolating, Purifying, and Characterizing Mycobacterium Bacteriophages Collected from Soil on the Winthrop University Campus

This was Winthrop University’s sixth year as part of the national HHMI sponsored SEA-PHAGES program. As freshman undergraduate students we isolated, purified, and characterized 4 bacteriophages found in the soil in and around Winthrop University, Rock Hill, South Carolina. COVID-19 limited the number of students in the laboratory at any given time, but working in pairs and eventually teams, we were able to isolate and culture four soil bacteriophages. Bacteriophages are viruses that infect bacterial host cells. They require a bacterial cell to reproduce and are specific to a particular host cell. Each phage was purified and amplified in the bacterial host Mycobacterium smegmatis mc2155. Following isolation, the phages were characterized following DNA extraction using restriction enzyme digests and gel electrophoresis. Calendula, Grindelwald, Allegro, and Moostard high-titer lysates were archived for future study. Two phages, Allegro and Moostard, were chosen to be sequenced at the University of Pittsburgh. The genome sequences will be returned to Winthrop for annotation. This research not only adds to the increasing knowledge of novel phages being discovered in this area of South Carolina but also allows Winthrop University and its students as members of an internationally renowned research program

Isolating, Purifying, and Characterizing Mycobacterium Bacteriophages Collected from Soil on the Winthrop University Campus

This was Winthrop University’s sixth year as part of the national HHMI sponsored SEA-PHAGES program. As freshman undergraduate students we isolated, purified, and characterized 4 bacteriophages found in the soil in and around Winthrop University, Rock Hill, South Carolina. COVID-19 limited the number of students in the laboratory at any given time, but working in pairs and eventually teams, we were able to isolate and culture four soil bacteriophages. Bacteriophages are viruses that infect bacterial host cells. They require a bacterial cell to reproduce and are specific to a particular host cell. Each phage was purified and amplified in the bacterial host Mycobacterium smegmatis mc2155. Following isolation, the phages were characterized following DNA extraction using restriction enzyme digests and gel electrophoresis. Calendula, Grindelwald, Allegro, and Moostard high-titer lysates were archived for future study. Two phages, Allegro and Moostard, were chosen to be sequenced at the University of Pittsburgh. The genome sequences will be returned to Winthrop for annotation. This research not only adds to the increasing knowledge of novel phages being discovered in this area of South Carolina but also allows Winthrop University and its students as members of an internationally renowned research program