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A laboratory course exploring the relationship between structure and function of yeast alcohol dehydrogenase 1 (ADH1)
Author(s) -
Huff Mary OˈMalley,
Plapp Bryce V.
Publication year - 2012
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.26.1_supplement.619.4
Subject(s) - yeast , biochemistry , enzyme , biology , alcohol dehydrogenase , computational biology
Project‐based (research‐driven) laboratory courses stimulate student involvement, improve critical thinking and initiate cooperative learning. To this end, a 10‐week laboratory project (or 7 sessions for a shorter version) was designed to accompany a second semester biochemistry course to reinforce the fundamental relationship between protein structure and function and cell physiology using yeast alcohol dehydrogenase I (ADH1). Working in small groups, students reviewed the scientific literature, proposed a specific amino acid change in the ADH1 sequence, and hypothesized how this change might alter the kinetics and structure of the enzyme. Host yeast expressing no ADH activity and a YEp13 shuttle vector containing the ADH1 gene were used. Students designed primers and performed site‐directed mutagenesis to generate a mutated form of ADH1. These plasmids were propagated in E. coli , sequenced, and reintroduced into yeast. Growth patterns on selective media were determined. Yeast cell lysates were separated by native gel electrophoresis for staining of enzyme activity and analysis of mobility changes. Kinetic values ( K m ) with ethanol and NAD as substrates were compared for mutated and wild type ADHs. For further analysis, ADH can be purified to homogeneity by chromatography and turnover numbers can be determined. Each group was required to submit a formal lab report resembling a journal publication. Student performance was evaluated based on the quality of the formal lab report, individual lab notebooks, and weekly participation. Funding to MOH from the TEAGLE Foundation.