Metabolic Characterization of Porcine Hepatocytes

In order to be approved for human trials, new drugs need to be tested on mammalian liver cells to determine toxicity. Currently, mouse and rat models are used with limited results because of their vastly different metabolisms. Model systems that utilize primate livers cells are going out of favor because of ethical issues. Cytotheryx Inc. is developing a porcine model system for use in drug trials that are metabolically similar to humans without the ethical concerns. In our research, we developed techniques to characterize porcine liver cell metabolism.

Analysis of a Putative Promoter in Mycobacteriophage JacoRen57

JacoRen57 is a cluster AB mycobacteriophage that infects Mycobacterium smegmatis mc²155. We recently reported on the characterization of a putative promoter in JacoRen57 using an mCherry reporter construct. This promoter is present in a gap upstream of a gene that is present in all AB phages. In all cases, these are forward genes immediately following a long series of reverse genes. The genes are most frequently identified as a RecA-like DNA recombinases but also as RepA by bioinformatics. To further analyze this putative promoter and gene product, NWC Molecular Genetics students cloned the RecA-like DNA recombinase into an E. coli expression vector with a TVMV removable N-terminal His-tag. They expressed and we purified the tagged protein and are using it to immunize Balb/c mice. We plan to use the antiserum to confirm RecA-like DNA recombinase expression patterns when JacoRen57 infects M. smegmatis

Investigating the Putative RecA-Like Recombinase Gene

Our Biochemistry: Molecular Genetics class has partnered with the Immunology class to investigate the expression of JacoRen57’s gene 50. The bacteriophage JacoRen57 – found in Sioux Center, Iowa (accession: MK279840). JacoRen57’s genome has sequenced by Pittsburg SEA-PHAGES Institute and fully annotated by Northwestern College students in 2018. A region between gene 49 and 50 caught our attention as there is a large gap between these genes. Almail et al., investigated if this is a transcription regulatory region for genes 49 and/or 50 (2021). This work demonstrated the region has a regulatory function in the direction of gene 50. Based on comparison genomics, gene 50 is a putative RecA-like recombinase (Almail et al., 2019). This protein has several functions including guiding the recombination of DNA within a gene. RecA-like recombinase allows the virus to evolve into new variants which can improve infection and replication. This is crucial for creating diversity in the genome and DNA repair mechanisms (Galletto and Kowalczykowski, 2007). To continue examination of gene 50 expression, we are working towards developing antibodies for this protein. To do this, the first step is to create an expression construct (Figure 1), express the protein in bacteria, purify the protein, and then use the purified protein to inoculate mice. This poster describes the construction of the expression vector. This work will provide valuable insight into the expression of gene 50, the RecA-like recombinase