Of all the TolC proteins we used in our project, three were ultimately submitted to the BioBrick registry. These submitted parts were tolC homologs from Yersenia pestis Angola (BBa_K1683000), Proteus mirabilis (BBa_K1683002), and Pseudomonas aeruginosa PA01 (oprM) (BBa_K168004). These proteins were submitted as a part of larger constructs which we used to express usable tolC molecules in our chassis organism, ΔtolC E. coli. These constructs consist of 4 major components. First is the pBAD promoter, an arabinose-induced strong E. coli promoter. Second is the strong E. coli ribosomal binding site (originally from BBa_B0034). Third is the E. coli tolC signaling sequence; this forms a chimeric protein with the tolC or tolC homolog of interest. See Project > Solution > TolC for more information on TolC.
These parts were characterized by western blotting and through Kirby-Bauer antibiotic assays. All three western blots showed increased protein expression with the inducer present. Yersenia pestis and Proteus mirabilis TolC proteins were even able efflux Novobiocin and Erythromycin as evidenced by the Kirby-Bauer assays. More detailed analysis is found on the Documentation page’s Results section.