Tunable Thermal Bioswitches for Noninvasive Genetic Regulation

Rapid advances in synthetic biology are driving the development of genetically engineered bacteria as microbial therapeutic and diagnostic agents for a host of human diseases. A critical capability for many envisioned in vivo applications is the ability to control the function of engineered bacteria in situ, as currently done using chemical inducers or visible light. However, systemic chemicals typically lack the spatial precision needed to control activity at specific anatomical sites, such as in the gastrointestinal tract or tumors, while optical approaches suffer from poor penetration in tissues. On the other hand, temperature can be controlled both globally and locally – at depth – using technologies such as focused ultrasound, infrared light and magnetic particle hyperthermia. In addition, body temperature can serve as an indicator of bacterial entry and exit from the host organism and the host’s condition. Given this potential, there are surprisingly few high-performance thermal bioswitches to control gene expression in engineered bacteria. The ideal bioswitch should have a sharp thermal transition resulting in a large change in activity (> 100-fold) over a few ºC, and its switching temperature should be tunable to enable a broad range of applications. Existing temperature-dependent regulators of gene expression fail to fulfill these criteria. To address this limitation, we have developed two families of tunable, orthogonal, temperature-dependent transcriptional repressors covering the biomedically relevant range of 32 to 46ºC.  These bioswitches feature sharp transitions (3-5ºC) and a large dynamic range (> 300-fold), superior to comparable protein and RNA-based regulators. The switching temperature can be tuned using a simple high-throughput screen, and orthogonality between the two bioswitch families enables multiplexed thermal control. We demonstrate the utility of engineered thermal bioswitches in two microbial therapy applications, first restricting the growth of engineered bacteria to body temperature to limit contagion outside the intended host, then using focused ultrasound to thermally activate engineered microbes at a specific targeted location. Given the multitude of ways that temperature can be controlled in situ, these tunable thermal bioswitches will enable a wide range of applications in bacterial synthetic biology and could also be adapted for use in eukaryotic systems.

D. Piraner and M. Abedi contributed equally to this work.