Tunable Temperature-Sensitive Transcriptional Activation Based on Lambda Repressor

Abstract

Temperature is a versatile input signal for the control of engineered cellular functions. Sharp induction of gene expression with heat has been established using bacteria- and phage-derived temperature-sensitive transcriptional repressors with tunable switching temperatures. However, few temperature-sensitive transcriptional activators have been reported that enable direct gene induction with cooling. Such activators would expand the application space for temperature control. In this technical note, we show that temperature-dependent versions of the Lambda phage repressor CI can serve as tunable cold-actuated transactivators. Natively, CI serves as both a repressor and activator of transcription. Previously, thermolabile mutants of CI, known as the TcI family, were used to repress the cognate promoters PR and PL. We hypothesized that TcI mutants can also serve as temperature-sensitive activators of transcription at CI’s natural PRM promoter, creating cold-inducible operons with a tunable response to temperature. Indeed, we demonstrate temperature-responsive activation by two variants of TcI with set points at 35.5 and 38.5 °C in E. coli . In addition, we show that TcI can serve as both an activator and a repressor of different genes in the same genetic circuit, leading to opposite thermal responses. Transcriptional activation by TcI expands the toolbox for control of cellular function using globally or locally applied thermal inputs.

Impact of exogenous aminoacyl-tRNA synthetase and tRNA on temperature sensitivity inEscherichia coli

“…Its heat-inducible nature may not align with the physiological conditions of bacteria. 42 Consequently, researchers have been exploring alternative cold-inducible switch systems, 41, 43, 44 although they remain notably rare.…”

Recent Synthetic Biology Approaches for Temperature- and Light-Controlled Gene Expression in Bacterial Hosts

“…As RNAT targets the late stage of gene expression, one can choose the RNAT system when a quick response is needed. Utilizing the ts-dimer module has advantages because of its relatively easy tunability at the working temperature [ 6 , 12 ] and modularity as a fusion partner, which broadens the scope of its applications. In contrast to the other ts modules, gene expression with the ts-replicon is irreversible.…”

“…In addition, the cI857 repression system is useful in constructing genetic circuits for bifunctional dynamic control [ 7 ] and kill switches [ 13 ]. cI857 variants can also be used for temperature-sensitive transcriptional activation [ 6 ]. Recently, a genetic circuit for a cold-inducible switch was developed [ 11 ].…”

“…From a synthetic biology perspective, nature’s mechanisms for sensing temperature and light have provided powerful insights for designing tools to achieve conditional gene expression. At the molecular level, researchers have utilized the heat shock response mechanism to construct temperature-sensitive gene expression systems such as the temperature-sensitive dimeric repressor [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 ], RNA polymerase (RNAP) [ 14 , 15 , 16 , 17 ], replication machinery [ 18 , 19 , 20 , 21 , 22 ], and oligonucleotide topology [ 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 ]. Photocaged [ 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 ] or photoswitchable [ 44 , 45 ] small-molecule inducers and photocaged transcription factors [ 46 ] have been used to develop light-driven gene expression systems.…”

Expanding the genetic programmability of Lactiplantibacillus plantarum

“…If phage-derived repressors prove to be effective in blocking the expression of the target gene in L. plantarum, they could be further modified to respond to external factors (e.g. temperature, light, chemicals), as it has previously been done for the Lambda phage repressor cI (Xiong et al, 2022). Moreover, bacteriophage-encoded RNA polymerases (Figure 4) are greatly used as synthetic biology tools and are frequently part of complex genetic circuits.…”