FIGURE 4.

Kanamycin Intein Splicing Reporter. For a splicing-dependent reporter, KanR interrupted with a catalytically active intein is resistant to kanamycin (A), while KanR interrupted with a catalytically inactive intein that cannot undergo splicing is not resistant to kanamycin (B). In (C), we show the experimental design to test the specific inhibition of protein splicing using a kanamycin-intein splicing reporter. In addition to cells containing the splicing-dependent KanR-intein fusion plasmid, control plasmids include KanR without an intein, a splicing inactive KanR-intein fusion, and kanamycin cells lacking a KanR-containing plasmid. In this design, serial dilutions of cells should be grown under four conditions: 1) without kanamycin and without splicing inhibitor; 2) without kanamycin and with splicing inhibitor; 3) with kanamycin and without splicing inhibitor; and 4) with kanamycin and with splicing inhibitor. Growth of the kanamycin sensitive and splicing inactive KanR-intein fusion strains should only occur in the absence kanamycin. Presence of the splicing inhibitor in the absence of kanamycin should not decrease growth of any strain. If inhibition of protein splicing occurs, the splicing-dependent KanR-intein fusion will display reduced survival compared to KanR without an intein. Interrupted, inactive exteins are colored gray, inteins are colored pink if active and gray if inactivated by mutation, and active KanR is colored blue.