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Tracking of Engineered Bacteria In Vivo Using Nonstandard Amino Acid Incorporation
journal contribution
posted on 2018-05-23, 00:00 authored by Pichet Praveschotinunt, Noémie-Manuelle Dorval Courchesne, Ilona den Hartog, Chaochen Lu, Jessica J. Kim, Peter Q. Nguyen, Neel S. JoshiThe rapidly growing
field of microbiome research presents a need
for better methods of monitoring gut microbes in vivo with high spatial and temporal resolution. We report a method of
tracking microbes in vivo within the gastrointestinal
tract by programming them to incorporate nonstandard amino acids (NSAA)
and labeling them via click chemistry. Using established
machinery constituting an orthogonal translation system (OTS), we
engineered Escherichia coli to incorporate p-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 via copper-free click reaction with a Cy5 dye conjugated to the dibenzocyclooctyl
(DBCO) group. We confirmed the functionality of the labeling strategy in vivo 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 in vivo tracking microbial strategies that may be compatible with noninvasive
imaging modalities and are capable of longitudinal spatiotemporal
monitoring of specific microbial populations.
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Engineered Bacteriain-frame UAG codongene encodingCy 5 dyemicrobiome researchmethodcopper-free click reactionmurine modelwork setscell surface proteinstrategyDBCOmonitoring gut microbesNSAAEscherichia colinoninvasive imaging modalitiesvivoNonstandard Aminoextracellular displaystrainpAzF incorporationspatiotemporal monitoringclick chemistrytractorthogonal translation systemOTS
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