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Semester‐long inquiry‐based molecular biology laboratory: Transcriptional regulation in yeast
Author(s) -
Oelkers Peter M.
Publication year - 2016
Publication title -
biochemistry and molecular biology education
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.34
H-Index - 39
eISSN - 1539-3429
pISSN - 1470-8175
DOI - 10.1002/bmb.21023
Subject(s) - plasmid , yeast , biology , agarose gel electrophoresis , saccharomyces cerevisiae , reporter gene , computational biology , gene , lysis , plasmid preparation , microbiology and biotechnology , genetics , gene expression , pbr322
A single semester molecular biology laboratory has been developed in which students design and execute a project examining transcriptional regulation in Saccharomyces cerevisiae . Three weeks of planning are allocated to developing a hypothesis through literature searches and use of bioinformatics. Common experimental plans address a cell process and how three genes that encode for proteins involved in that process are transcriptionally regulated in response to changing environmental conditions. Planning includes designing oligonucleotides to amplify the putative promoters of the three genes of interest. After the PCR, each product is cloned proximal to β‐galactosidase in a yeast reporter plasmid. Techniques used include agarose electrophoresis, extraction of DNA from agarose, plasmid purification from bacteria, restriction digestion, ligation, and bacterial transformation. This promoter/reporter plasmid is then transformed into yeast. Transformed yeast are cultured in conditions prescribed in the experimental design, lysed and β‐galactosidase activity is measured. The course provides an independent research experience in a group setting. Notebooks are maintained on‐line with regular feedback. Projects culminate with the presentation of a poster worth 60% of the grade. Over the last three years, about 65% of students met expectations for experimental design, data acquisition, and analysis. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(2):145–151, 2017.