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. Author manuscript; available in PMC: 2020 Apr 17.
Published in final edited form as: J Am Chem Soc. 2019 Apr 8;141(15):6204–6212. doi: 10.1021/jacs.8b12954

Figure 3.

Figure 3.

A dual ncAA incorporation system using EcTrp(UCA) and Pyl(CUA) pairs in ATMW1 E. coli. A) Scheme of the dual ncAA incorporation experiment. B) Expression of sfGFP-3UGA-151UAG using this system in the presence of indicated ncAA substrates, measured by its characteristic in-cell fluorescence. C) Purified sfGFP-3-5HTP-151-CpK has the expected mass (top panel). Treatment with 4CDZ and tetrazine-fluorescein results in its one-pot quantitative double labeling, and confirms the feasibility of using CRACR and SPIEDAC for protein modification at two different sites.