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Purpose In the programming of tumor-targeting bacteria, various therapeutic or reporter genes are expressed by different gene-triggering strategies. Previously, we engineered pJL87 plasmid with an inducible bacterial drug delivery system that simultaneously co-expressed two genes for therapy and imaging by a bidirectional  tet  promoter system only in response to the administration of exogenous doxycycline (Doxy). In this multi-cassette expression approach,  tetA  promoter (P  tetA  ) was 100-fold higher in expression strength than  tetR  promoter (P  tetR  ). In the present study, we developed pJH18 plasmid with novel Doxy-inducible gene expression system based on a  tet  promoter. Procedures In this system, Tet repressor (TetR) expressed by a weak constitutive promoter binds to  tetO  operator, resulting in the tight repression of gene expressions by P  tetA   and P  tetR  , and Doxy releases TetR from  tetO  to de-repress P  tetA   and P  tetR  . Results In  Salmonella  transformed with pJH18, the expression balance of bidirectional  tet  promoters in pJH18 was remarkably improved (P  tetA  :P  tetR   = 4~6:1) compared with that of pJL87 (P  tetA  :P  tetR   = 100:1) in the presence of Doxy. Also, the expression level by novel  tet  system was much higher in  Salmonella  transformed with pJH18 than in those with pJL87 (80-fold in  rluc8  and 5-fold in  clyA ). Interestingly, pJH18 of the transformed  Salmonella  was much more stably maintained than pJL87 in antibiotic-free tumor-bearing mice (about 41-fold), because only pJH18 carries  bom  sequence with an essential role in preventing the plasmid-free population of programmed  Salmonella  from undergoing cell division. Conclusions Overall, doxycycline-induced co-expression of two proteins at similar expression levels, we exploited bioluminescence reporter proteins with preclinical but no clinical utility. Future validation with clinically compatible reporter systems, for example, suitable for radionuclide imaging, is necessary to develop this system further towards potential clinical application.

Optimized Doxycycline-Inducible Gene Expression System for Genetic Programming of Tumor-Targeting Bacteria