General Strains and Plasmid Constructions

Natural amino acids and ncAAs were purchased from Chem Impex Inc (Wood Dale, IL, United States). The ncAA SeCbzK (10) (Wang Z.U. et al., 2012) and CbzKOH (11) (Li et al., 2012; Wang, 2012) (Scheme 1) were synthesized by the reported methods. PCR was performed using the KOD hot start polymerase kit (Merck). Oligonucleotide synthesis and DNA sequencing were done by Genomics Inc. (Taipei, Taiwan). The primer sequences are listed in the Supplementary Materials. Construction of the pET-pylT-sfGFP-TAG2 and the pET-pylT-sfGFP-TAG27 plasmids followed general cloning protocols. The sfGFP-TAG2 gene was amplified by PCR from pET-pylT-sfGFP (Guo et al., 2014) with the primers sfGFP-TAG2-NdeI-F and sfGFP-SacI-R2. The sfGFP-TAG27 was generated by overlap extension PCR with two fragments (fragment 1: primer sfGFP-NdeI-F1 and sfGFP-TAG27-R; fragment 2: sfGFP-TAG27-F and sfGFP-SacI-R2). The overlapped sfGFP gene products and pET-pylT plasmid were then double-digested with restriction enzyme NdeI and SacI, and then T4 DNA ligase was used for DNA ligation.

In the construction of the plasmid pCDF-N-PylRS variants, the PylRS-R61K/H63Y/S193R (N-PylRS) gene was generated by overlap extension PCR with three fragments using the template pCDF-PylRS: fragment 1: PylRS-NcoI-F and PylRS-R61K/H63Y-R; fragment 2: PylRS-R61K/H63Y-F and PylRS-S193R-R; fragment 3: PylRS-S193R-F and PylRS-BamHI-R. In preparing pCDF-PylRS-R61K, pCDF-PylRS-H63Y, and pCDF-PylRS-S193R, the same procedures were performed with the following primers to produce PylRS-R61K, PylRS- H63Y, and PylRS-S193R genes with pCDF-PylRS template and following the overlap extension PCR; PylRS-R61K gene: fragment 1, PylRS-NcoI-F and PylRS-R61K-R; fragment 2, PylRS-R61K-F and PylRS-BamHI-R; PylRS-H63Y gene: fragment 1, PylRS-NcoI-F and PylRS-H63Y-R; fragment 2, PylRS-H63Y-F and PylRS-BamHI-R; PylRS-S193R gene: fragment 1, PylRS-NcoI-F and PylRS-S193R-R; fragment 2, PylRS-S193R-F and PylRS-BamHI-R. In preparing pCDF-PylRS-ND, the PylRS-ND gene fragment was generated by PCR with two primers, PylRS-NcoI-F and PylRS-P149-BamHI-R, and pCDF-PylRS was used as a template. In the preparation of pCDF-PylRS-D1, the spacer sequence (TGAAAAAAGCGATG), including the TGA stop codon and the ATG start codon, was installed between positions P149 and A150 of the PylRS gene. The PylRS-D1 gene fragment was generated by overlap extension PCR with two fragments using pCDF-PylRS as a template. Fragment 1 was generated by the primers PylRS-NcoI-F and PylRS-P149SSS-R1; fragment 2 was generated by the primers PylRS-P149SSS-F2 and PylRS-BamHI-R. Different lengths of linker were installed in PylRS variants between sposition 149 and 150. In preparing pCDF-PylRS-L1, pCDF-PylRS-L2, and pCDF-PylRS-L3 (Figure 1B), the same procedures were performed by the following primers to produce PylRS-L1, PylRS-L2, and PylRS-L3 genes with pCDF-PylRS template and following overlap extension PCR: PylRS-L1 gene, PylRS-NcoI-F, and Linker-1XG4S-R1, Linker-1XG4S-F2, and PylRS-BamHI-R; PylRS-L2 gene: fragment 1, PylRS-NcoI-F and Linker-2XG4S-R1; fragment 2, Linker-2XG4S-F2 and Linker-2XG4S-R2; fragment 3, Linker-2XG4S-F3 and PylRS-BamHI-R; PylRS-L3 gene: fragments 1 and 3 were from the PylRS-L2 gene; fragment 2, Linker-3XG4S-F2 and Linker-3XG4S-R2.

The pCDF-N-PylRS plasmids with the inserted linker, pCDF-N-PylRS-D1, pCDF-N-PylRS-L1, pCDF-N-PylRS-L2, and pCDF-N-PylRS-L3 (Figure 1B), were then constructed using the procedures mentioned above. pCDF-ZRS was derived from pEVOL-mKRS1-pylT (Wang Z.U. et al., 2012). The N-PylRS mutations were transplanted to ZRS by the methods mentioned earlier to construct pCDF-N-ZRS. The linkers were inserted into ZRS to generate pCDF-N-ZRS-D1, pCDF-ZRS-L1, pCDF-ZRS-L2, and pCDF-ZRS-L3 plasmids. All the ligated products were transformed to E. coli DH5α, and the colonies were selected for DNA sequencing, respectively.