Expanding the Toolbox for Thermal Control of E. coli: Cold-Activated Transcription with Applications in Temperature Self-Regulation

Temperature can be used to control engineered E. coli — for example, the living component of an engineered living material (ELM) - through the use of thermolabile transcription factors. Sharp induction of gene expression with heat has been established using these bacteria- and phage-derived proteins. Here, we expand the toolbox for thermal control of E. coli through both direct cold-induced gene expression and through the construction of genetic circuits to invert heat-induced gene expression. We accomplish direct induction at low temperatures through the use of temperature-sensitive mutants of Lambda repressor as transcriptional activators. In addition, we show that a temperature-sensitive mutant of Lambda repressor can serve as an activator and a repressor of different genes simultaneously in one genetic circuit, leading to opposite thermal responses and serving as a temperature switch. Next, we demonstrate inversion of a temperature-sensitive repressor using a temperature insensitive repressor. We apply this multicomponent switch to engineer a temperature self-regulation circuit for E. coli-based ELMs. Seasonal variation in ambient temperature presents a challenge in deploying ELMs outside of a laboratory environment, because E. coli growth rate is impaired both below and above 37°C. Our construct enables E. coli to produce a light-absorptive pigment in response to environmental temperature below 36°C with the goal of allowing the cells to absorb sunlight and locally warm to their optimal growth temperature. We demonstrate the efficacy of our pigment temperature switch in a model flat ELM growing at 32°C and 42°C in a home-built illuminated growth chamber. Below 36°C, our engineered E. coli increase in pigmentation, causing an increase in sample temperature and growth rate above non-pigmented bacteria. On the other hand, above 36°C, they decrease in pigmentation, protecting their growth compared to bacteria with temperature- independent high pigmentation. Integrating our temperature homeostasis circuit into an ELM has the potential to improve ELM performance by optimizing growth and protein production in the face of seasonal temperature changes.