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. 2007 Dec 14;190(4):1298–1307. doi: 10.1128/JB.01639-07

FIG. 2.

FIG. 2.

Construction of the suppressor tRNA shuttle plasmids. (A) An amber stop codon (TAG) was introduced into the ampicillin resistance gene (bla) of the pPM13 shuttle vector to create the plasmid pSM25. P13, P. haloplanktis TAC125 P13 promoter; oriT, origin of conjugative transfer; oriR, origin of replication (P. haloplanktis); oriC, origin of replication (E. coli). (B) A selectable marker, the chloramphenicol resistance gene (cat) of pSU18, was amplified by PCR and cloned into the pSM25 plasmid between the NdeI and XbaI restriction sites to generate plasmid pSM27. (C) The PvuII fragments of amber suppressor plasmids (Interchange Amber Suppressor in vivo Mutagenesis System kit; Promega) carrying suppressor tRNA genes with a promoter and a transcription terminator were inserted into the SmaI restriction site of pSM27, generating the suppressor tRNA shuttle plasmids listed in the table. The arrows show the orientations of the cloned fragments. P, synthetic promoter derived from lipoprotein gene lpp; T, synthetic transcription terminator based on rRNA operon rrnC. See the text and Table 1 for details.