A tuneable genetic switch for tight control of tac promoters in Escherichia coli boosts expression of synthetic injectisomesAlejandro Asensio‐Calavia, Álvaro Ceballos‐Munuera, Almudena Méndez‐Pérez, Beatriz Álvarez, Luis Ángel Fernández
- Applied Microbiology and Biotechnology
Biosafety of engineered bacteria as living therapeutics requires a tight regulation to control the specific delivery of protein effectors, maintaining minimum leakiness in the uninduced (OFF) state and efficient expression in the induced (ON) state. Here, we report a three repressors (3R) genetic circuit that tightly regulates the expression of multiple tac promoters (Ptac) integrated in the chromosome of E. coli and drives the expression of a complex type III secretion system injectisome for therapeutic protein delivery. The 3R genetic switch is based on the tetracycline repressor (TetR), the non‐inducible lambda repressor cI (ind‐) and a mutant lac repressor (LacIW220F) with higher activity. The 3R switch was optimized with different protein translation and degradation signals that control the levels of LacIW220F. We demonstrate the ability of an optimized switch to fully repress the strong leakiness of the Ptac promoters in the OFF state while triggering their efficient activation in the ON state with anhydrotetracycline (aTc), an inducer suitable for in vivo use. The implementation of the optimized 3R switch in the engineered synthetic injector E. coli (SIEC) strain boosts expression of injectisomes upon aTc induction, while maintaining a silent OFF state that preserves normal growth in the absence of the inducer. Since Ptac is a commonly used promoter, the 3R switch may have multiple applications for tight control of protein expression in E. coli. In addition, the modularity of the 3R switch may enable its tuning for the control of Ptac promoters with different inducers.