The pDule plasmid contains genes that encode the ncAA-RS and the tRNACUA, an origin of replication (must be compatible with the pBAD origin of replication), and a gene that encodes TetA protein (which confers tetracycline resistance). These plasmids must both be transformed into the cell in order for full-length, ncAA-containing protein to be produced. To verify that the cells contain both plasmids before initiating overexpression, the cells are grown in the presence of the antibiotics ampicillin and tetracycline, ensuring that only cells that contain both plasmids will be able to grow in the media.
