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.