Title of Abstract

Characterizing the Effect of Bacteriophage Cain Genes on Mycobacterium smegmatis

Poster Number

38

Session Title

Poster Session 2

Faculty Sponsor (for work done with a non-Winthrop mentor)

Kathryn Kohl, Ph.D.

College

College of Arts and Sciences

Department

Biology

Abstract

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.

Start Date

15-4-2022 12:00 PM

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Apr 15th, 12:00 PM

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.