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ABSTRACT Nicotine is a major alkaloid in tobacco plants and an addictive component of tobacco products. Some bacteria grow on tobacco plants and have evolved the ability to metabolize nicotine. As part of our microbiology teaching lab, we used minimal media with nicotine as the sole carbon source to isolate nicotine-degrading bacteria from tobacco leaves and commercial tobacco products. Students then identified these bacteria using 16S rRNA sequencing and biochemical assays and assessed their ability to catabolize nicotine using UV spectroscopy. Students were able to isolate and identify 14 distinct genera that can metabolize nicotine. This modification of the commonly used unknown project gave students firsthand experience using selective media, and students got the opportunity to work with largely uncharacterized microbes with a real-world connection to public health, which increased student engagement. Students had the opportunity to think critically about why nicotine-degrading microorganisms associate with tobacco plants, why there are different bacteria that use the same specialized metabolism, and how these organisms are isolated from other bacteria using selective media.