10.1021/acssynbio.8b00135.s001 Pichet Praveschotinunt Pichet Praveschotinunt Noémie-Manuelle Dorval Courchesne Noémie-Manuelle Dorval Courchesne Ilona den Hartog Ilona den Hartog Chaochen Lu Chaochen Lu Jessica J. Kim Jessica J. Kim Peter Q. Nguyen Peter Q. Nguyen Neel S. Joshi Neel S. Joshi Tracking of Engineered Bacteria <i>In Vivo</i> Using Nonstandard Amino Acid Incorporation American Chemical Society 2018 Engineered Bacteria in-frame UAG codon gene encoding Cy 5 dye microbiome research method copper-free click reaction murine model work sets cell surface protein strategy DBCO monitoring gut microbes NSAA Escherichia coli noninvasive imaging modalities vivo Nonstandard Amino extracellular display strain pAzF incorporation spatiotemporal monitoring click chemistry tract orthogonal translation system OTS 2018-05-23 00:00:00 Journal contribution https://acs.figshare.com/articles/journal_contribution/Tracking_of_Engineered_Bacteria_i_In_Vivo_i_Using_Nonstandard_Amino_Acid_Incorporation/6450278 The rapidly growing field of microbiome research presents a need for better methods of monitoring gut microbes <i>in vivo</i> with high spatial and temporal resolution. We report a method of tracking microbes <i>in vivo</i> within the gastrointestinal tract by programming them to incorporate nonstandard amino acids (NSAA) and labeling them <i>via</i> click chemistry. Using established machinery constituting an orthogonal translation system (OTS), we engineered <i>Escherichia coli</i> to incorporate <i>p</i>-azido-l-phenylalanine (pAzF) in place of the UAG (amber) stop codon. We also introduced a mutant gene encoding for a cell surface protein (CsgA) that was altered to contain an in-frame UAG codon. After pAzF incorporation and extracellular display, the engineered strains could be covalently labeled <i>via</i> copper-free click reaction with a Cy5 dye conjugated to the dibenzocyclooctyl (DBCO) group. We confirmed the functionality of the labeling strategy <i>in vivo</i> using a murine model. Labeling of the engineered strain could be observed using oral administration of the dye to mice several days after colonization of the gastrointestinal tract. This work sets the foundation for the development of <i>in vivo</i> tracking microbial strategies that may be compatible with noninvasive imaging modalities and are capable of longitudinal spatiotemporal monitoring of specific microbial populations.