Discovery-oriented teaching: The use of chimeric proteorhodopsins for the development of a lab curriculum in marine microbiology and for the discovery of natural red-shifted proteorhodopsins

Student laboratory courses in microbial ecology are conducted as condensed courses, where theory and wet lab work are combined in a very intensive short time period. During the last decades, the study of marine ecology became molecular-based, most of the research requiring sequencing that is often not available at the course facilities, and takes more time than the course allows. Therefore, students often find themselves obtaining and analyzing results weeks after the course ended. In this work, we describe a protocol combining molecular and functional methods for analyzing microbial rhodopsins, with visible results in only 4-5 days. We created a chimeric plasmid suitable for direct expression of environmentally retrieved proteorhodopsins (PRs) using PCR, and cloned it into Escherichia coli cells for visualization and functional analysis. Using this assay, we discovered several exceptional cases of PRs whose phenotype is different than predicted based on sequence only. We expect this assay to improve the marine microbiology laboratories experience for students, gaining fast feedback and reward for their work, and to promote the use of functional assays for discovery of marine diversity that was masked by sequence-based methods.