Construction of a stably integrated mammalian toggle switch from bacterial components*

Synthetic gene circuits allow for regulation of  cellular properties, such as cell state. A genetic toggle switch, built originally in bacteria, is one such example, composed of two mutually repressive genes modulated by inducers. Previous designs in mammalian cells have not been stably, chromosomally integrated, only transiently expressed, and have used mammalian components, posing issues in terms of off target effects as well as longer activation times. Considering that stable chromosomal integration is needed for long-term applications, such as therapeutics, we redesigned the mammalian toggle switch with bacterial components.  This chromosomally integrated design uses the TetR and PhlF bacterial repressor proteins to compete with RNA polymerase for promoter binding, for faster and more specific activity. The inducers doxycycline (Dox) and 2,4-diacetylphloroglucinol (DAPG), bind TetR and PhlF, respectively, and relieve repression of downstream protein expression. Initial induction experiments showed that the circuit was responsive to Dox, but not DAPG, likely due to cellular  uptake issues with DAPG. Mathematical modeling suggested solutions to overcome this issue. Liposomal delivery of Dox and DAPG to improved induction efficiency. This redesigned mammalian toggle switch will contribute to the growing number of synthetic biochemical tools, with strong potential for cellular control, therapeutics and diagnostics.