Postdoctoral Fellowship: High-resolution imaging of starch utilization proteins in live human gut bacteria using small fluorescent tags
Bacteroides thetaiotaomicron is one of the most abundant inhabitants of the mammalian gut. This obligate anaerobe is capable of degrading numerous carbohydrates, including those that are resistant to digestion by the host. This process occurs via substrate-specific polysaccharide utilization systems. The best studied of these is the starch utilization system (Sus). The aim of this research is to label the Sus proteins with fluorescent unnatural amino acids (UAAs). The small size of these UAAs means that they are minimally disruptive to the labeled protein and are conducive to live-cell imaging. Strains with two labeled Sus proteins will be constructed, and single-molecule fluorescence microscopy will be employed to observe and quantify the localization and dynamics of individual proteins and protein complexes on the nanometer scale.
This research will improve understanding of the role of starch breakdown by symbiotic bacteria during normal human digestion, as well as the process of carbohydrate metabolism by bacteria in general. The main challenge in this proposal will be adapting fluorescent amino acids for use in a non-model system and for investigation by single-molecule fluorescence microscopy. Success would demonstrate that the promising technique of fluorescent amino acid incorporation into living organisms is truly generalizable. This project will also provide a platform for outreach through the UM Women in Science and Engineering (WISE) Girls in Science and Engineering (GISE) summer camp. Students at the camp will receive hands-on laser and microscopy demonstrations and learn to use these tools to examine the behavior of proteins inside cells.
