hygromycin-a and indigoidine

To build or dissect complex pathways in bacteria and mammalian cells, it is often necessary to recur to at least two plasmids, for instance harboring orthogonal inducible promoters. Here we present SiMPl, a method based on rationally designed split enzymes and intein-mediated protein trans-splicing, allowing the selection of cells carrying two plasmids with a single antibiotic. We show that, compared to the traditional method based on two antibiotics, SiMPl increases the production of the antimicrobial non-ribosomal peptide indigoidine and the non-proteinogenic aromatic amino acid para-amino-L-phenylalanine from bacteria. Using a human T cell line, we employ SiMPl to obtain a highly pure population of cells double positive for the two chains of the T cell receptor, TCRα and TCRβ, using a single antibiotic. SiMPl has profound implications for metabolic engineering and for constructing complex synthetic circuits in bacteria and mammalian cells.

Topics: Ampicillin Resistance; Anti-Bacterial Agents; Bacteria; Cell Line; Chloramphenicol Resistance; Cinnamates; Drug Resistance, Bacterial; Humans; Hygromycin B; Inteins; Metabolic Engineering; Piperidones; Plasmids; Protein Splicing; Puromycin; Receptors, Antigen, T-Cell, alpha-beta; T-Lymphocytes; Trans-Splicing