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RNAse One Cloning, Expression and Enzyme Analysis Provides Research Sequence for Biochemistry Laboratory Course
Author(s) -
Bailey Cheryl P
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.574.6
Subject(s) - rnase p , cloning (programming) , biochemistry , biology , microbiology and biotechnology , sequence analysis , sequence (biology) , computational biology , rna , gene , computer science , programming language
Students in a biochemistry laboratory course were posed with the question of how they would design experiments that included a protein of interest. Isolation and characterization of the protein quickly came to the forefront, which directed conversation to why bacterially expressed tagged proteins are so prevalently used in research laboratories. Students utilized BLAST to search for E. coli RNAse One sequence and designed primers PCR to isolate the coding sequence of RNAse One followed by coding sequence for Glutathione‐S‐Transferase (GST). Successful PCR products were cloned into the pGEX™ expression vector. Expressed protein was purified with MagneGST™ beads and protein concentration measured. Total RNA was isolated from Xenopus oocytes and subjected to degradation by purified RNAse One. RNAse One activity was compared to RNAse A and RNAse H with purchased substrates. Literature searches revealed potential further tests with inhibitors. This experience culminated in a poster session with campus faculty invited to participate and ask “how?”, “why?” and “what now?” questions.