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. Author manuscript; available in PMC: 2023 Apr 30.
Published in final edited form as: J Mol Biol. 2021 Oct 13;434(8):167304. doi: 10.1016/j.jmb.2021.167304

Figure 3.

Figure 3.

Development of an optimized pEVOL suppressor plasmid. a) Vector map of the first-generation pEVOL vector (full sequence provided in SI). (b) A scheme showing the components of the vector which were altered for optimization: p15A, RSF, or ColA ori (light blue); tacI or glnS promoter (navy blue); EcTrpRS-h13 or -h14 (gray); proK, lpp, or leuV promoter (navy blue). (c) Expression of sfGFP-151-TGA using pEVOL vectors (proK-tRNA and tacI-TrpRS) with varying oris in ATMW-BL21 in the presence or absence of 5HTP. (d) Expression of sfGFP-151-TGA using pEVOL vectors containing either an inducible tacI or constitutive glnS promoter on the EcTrpRS (tRNA expressed from proK). (e) Expression of sfGFP-151-TGA using pEVOL vectors with different combinations of EcTrpRS and tRNAEcTrpUCA promoters (tacI/glnS = EcTrpRS promoter; leuV/lpp = tRNAEcTrpUCA promoter). (f) MS analysis of the sfGFP-151-TGA reporter protein confirming 5HTP incorporation using pEVOL-tacI-EcTrpRS-h13-leuV-tRNA. Full MS spectrum is provided in Supplementary Figure 2. (g) SDS-PAGE analysis of purified sfGFP-151– 5HTP and sfGFP-WT. Full SDS-PAGE gel is provided in Supplementary Figure 3. For all sfGFP reporter expression analyses, its characteristic fluorescence was measured in resuspended cells and normalized to OD600.