We have continued and plan to expand the guided inquiry research component of our one semester Biochemistry Laboratory Course (BIOC463A). Since 2009, the second portion of the semester involved a Special Research Project. Initially our studies involved examination of the structural importance and roles of two disulfide bonds in alkaline phosphatase, a periplasmic enzyme from E. coli, using the chemical reducing agents beta-mercaptoethanol, dithiothreitol, and triscarboxyethylphosphine. These early studies included a variety of spectroscopic methods including activity assays, fluorescence spectroscopy, and circular dichroism (CD) spectroscopy.
In 2013 we began a two year plan to develop our own protein expression system, which in fall 2014 was realized when we designed our current pETHSUL-AP vector that has successfully incorporated into Origami cells (a special strain of E. coli developed by Stratagene to induce disulfide bonds in the expressed proteins). In the spring 2015 semester we designed our first set of site-directed mutants of AP using PCR mutagenesis techniques, which is also being done by the current fall 2015 sections of BIOC463A. Our most recent work from the spring 2016 semester has helped us quantitatively assess the activity of our wild type and mutant enzymes from the Origami cells compared to commercially available sources.
Our attempts at incorporating real research into our course has been highlighted in four posters presented at the American Society for Biochemistry and Molecular Biology (ASBMB) annual meetings as well as two invited talks describing our transformation process.