1. Volume 24, Issue 8, Pages 1048-1055.e3 (August 2017)
Rapid Screening and Identification of Living Pathogenic Organisms via Optimized Bioorthogonal Non-canonical Amino Acid Tagging 
Allison Rae Sherratt, Yanouchka Rouleau, Christian Luebbert, Miroslava Strmiskova, Teodor Veres, Sabah Bidawid, Nathalie Corneau, John Paul Pezacki 
Cell Chemical Biology 
Volume 24, Issue 8, Pages e3 (August 2017)
DOI: /j.chembiol
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2. Cell Chemical Biology 2017 24, 1048-1055. e3DOI: (10. 1016/j. chembiol
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3. Figure 1
Surface-Labeling-Optimized BONCAT for the Capture of Living Bacteria
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4. Figure 2
Metabolic Labeling of Newly Synthesized Proteins in Gram-Negative and Gram-Positive Bacteria
(A and B) BL21 E. coli (A) or L. innocua (B) were cultured in the presence of methionine (top panels) or homopropargylglycine (HPG, bottom panels), as well as methionine biosynthesis inhibitory amino acids (lysine, threonine, phenylalanine, isoleucine, leucine, and valine) overnight. Cultures were then washed in PBS, reacted with 50 μM Alexa 488-azide by CuAAC, and detected by fluorescence microscopy. Bright field is displayed on the left and fluorescence is on the right. Fluorescence levels are adjusted above the background detected for cells grown in the presence of methionine.
(C) HPG is incorporated into newly synthesized proteins. BL21 E. coli were cultured for 30 min with the indicated concentration of HPG, methionine, or chloramphenicol (pre-incubated for 10 min) in the presence of methionine biosynthesis inhibitory amino acids. Bacteria cell lysates were reacted with rhodamine-azide by CuAAC and analyzed by in-gel fluorescence. Scale bars, 5 μm. See also Figures S1–S3.
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5. Figure 3
Capture of Translationally Active E. coli Bacteria via Surface-Labeling-Optimized BONCAT
BL21 E. coli were cultured in the presence of methionine or HPG, as well as methionine biosynthesis inhibitory amino acids overnight, reacted with biotin-azide by CuAAC, and either detected by fluorescence microscopy after incubation with Cy3-streptavidin (top panels, bright field on left), or captured after incubation with magnetic M-280 streptavidin-coated Dynabeads (bottom panels, red arrows). Scale bars, 5 μm. See also Figure S4.
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6. Figure 4 Combined Immunomagnetic Capture and BONCAT of E. coli
K12 E. coli were cultured for 1 hr in the presence of methionine or HPG, as well as methionine biosynthesis inhibitory amino acids and streptavidin-coated magnetic beads treated with PBS or biotinylated E. coli antibody. The beads were washed in PBS, reacted with Alexa 488-azide by CuAAC, and detected by fluorescence microscopy. Bright field is displayed on the left and fluorescence is on the right. Fluorescence levels are adjusted above the background detected for cells grown in the presence of methionine. Scale bars, 5 μm. See also Figure S4.
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7. Figure 5
Metabolic Labeling and Serotype Identification of Living Verotoxigenic E. coli
(A) Non-canonical amino acid incorporation and fluorescence labeling of VTEC. VTEC strains with serotypes O26:H11, O45:H2, O103:H2 were cultured in the presence of methionine or HPG, as well as methionine biosynthesis inhibitory amino acids (lysine, threonine, phenylalanine, isoleucine, leucine, and valine) overnight, reacted with Alexa 488-azide via CuAAC, and detected by fluorescence microscopy. Bright field is shown on the left; fluorescence is shown on the right for each condition. Fluorescence is displayed above the background levels of each paired methionine sample (Figure S6).
(B) Metabolic labeling and click chemistry of living VTEC does not interfere with downstream strain identification by PCR. Strain-specific PCR products were amplified from VTEC strains (O45:H2, 255bp; O103:H2, 205bp; O26:H11, 283bp) and detected by agarose gel, after being cultured in the presence of methionine or HPG, then reacted with Alexa 488-azide by CuAAC. Scale bars, 10 μm. See also Figures S5 and S6.
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8. Figure 6
Dual Metabolic Label Incorporation, Fluorescence Labeling, and Capture of Living E. coli
BL21 E. coli were cultured in the presence of methionine and KDO or HPG and KDO-azide, as well as methionine biosynthesis inhibitory amino acids (lysine, threonine, phenylalanine, isoleucine, leucine, and valine) overnight. The cells were first reacted with biotin-azide to biotinylate surface proteins, then reacted with Alexa 488-alkyne to fluorescently label LPS molecules. Surface biotinylation was either fluorescently detected after incubation with Cy3-streptavidin (A), or bacteria were captured after incubation with streptavidin-coated magnetic beads (B). Fluorescence levels are adjusted above the background detected for cells grown in the presence of KDO and methionine. Scale bars, 5 μm.
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