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. 2018 Sep 28;8:14527. doi: 10.1038/s41598-018-32824-1

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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(A) Schematic representation of plasmids carrying different number of encoded PylT genes and EGFP^150TAG-HA. Plasmid a, with no encoded tRNA served as control, while plasmid b carries a single copy of PylT. Plasmids c and d carry two encoded copies of PylT, either cloned separately (plasmid c, marked 1 + 1) or in tandem (plasmid d, marked 2). Plasmid e carries 4 copies of PylT in two cassettes, each containing two PylT genes cloned in tandem (marked 2 + 2). One set of plasmids was cloned with wild-type Pyl-RS and a second set was cloned with evolved BCN-RS (a total of 10 plasmids). (B) Chemical structures of the amino acids used in this study: Boc-Lys (1) and BCN-Lys (2).