Joshua is an up-and-coming senior at Bellarmine University in Louisville, Kentucky. This summer he is working with Dr. Addepalli and is focused on cloning and expressing the novel ribonuclease genes CCRN2 and Angiogenin in E. coli cells, which would allow for the characterization of the proteins. The genes of interest will be ligated into a piece of PET22B plasmid, resulting in a recombinant vector. The recombinant vector will be transferred to a host bacterium, competent BL21 E. coli cells, and plated onto LB agar plates. The E. coli cell colonies that grow onto the LB agar plate will be screened for recombinant plasmid that contain the gene of interest and will subsequently be induced to express the ribonuclease gene so the expressed protein could be purified. The purified protein will be tested for its ribonuclease activity, cleavage specificity through testing with a known sequence of RNA, and subjected to liquid chromatography-mass spectrometry (LC-MS) analysis for characterization of oligonucleotide digestion products. Through this analysis, the RNA cleavage behavior of the ribonuclease will be identified.
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