Lealia Xiong

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Tunable temperature-sensitive transcriptional activation based on lambda repressor

Lealia L. Xiong, Michael A. Garrett, Marjorie T. Buss, Julia A. Kornfield, and Mikhail G. Shapiro in ACS Synthetic Biology

Graphical abstract. Cartoon of a green bacterium transforming into a magenta bacterium with an increase in temperature. The bacteria have schematics of genetic circuits inside.

Abstract | Figure 1 | Figure 2 | Data tools

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.

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Figure 1

TcI mutants act as tunable, temperature-sensitive transactivators.

We constructed a model gene circuit to characterize the ability to activate transcription of two mutants, TcI38 and TcI39, previously developed as repressors:

Gene circuit diagrams of TcI variants.

TcIx (x = 38, 39), wildtype CI, or no activator activates expression of mWasabi (GFP) from the PRM promoter.

We quantified the gene expression level controlled by TcI38 or TcI39 via GFP fluorescence measured by flow cytometry and compared with the level driven by wildtype CI or without an activator in E. coli.

Summed frequency histograms for GFP channel for expression of GFP from PRM promoter by TcI38, TcI39, wildtype cI, or at baseline (no activator). NF indicates nonfluorescent control measured in the same channel:

Thermal profile of mean population fluorescence of GFP expressed from the PRM promoter with activation by TcI38, TcI39, and wildtype CI, or at baseline (no activator), calculated from geometric means of flow cytometry data:

Mean fluorescence expressed by TcI variants at temperatures ranging from 32 to 42 Celsius.

Thermal profile of % wildtype activation of gene expression by TcI38 and TcI39:

TcI variants as a percentage of wildtype activation at temperatures ranging from 32 to 42 Celsius.

At each temperature, 100% wildtype activation indicates expression equal to wildtype CI, and 0% activation indicates expression equal to unactivated PRM.

Figure 2

TcI39 simultaneously activates and represses, serving as a temperature-controlled state switch.

We assembled a construct wherein expression of GFP from the PRM promoter is activated by TcI39 and expression of mRFP1 (RFP) from the PR–PL tandem promoter is repressed by TcI39:

Gene circuit diagram of TcI39 state switch with state of regulation arcs at low and high temperature.

We assayed the thermal response of this genetic circuit in E. colivia GFP and RFP fluorescence measured by flow cytometry.

Bivariate kernel density estimation for RFP channel and GFP channel:

Marginal plots: summed frequency histograms for RFP channel (right) and GFP channel (top). NF indicates nonfluorescent control measured in each channel (not shown in central plot for visual clarity).

Thermal profile of mean population fluorescence of GFP and RFP expressed in E. coli containing the TcI39 switch construct:

Mean red and green fluorescence of TcI39 state switch at temperatures ranging from 32 to 42 Celsius.

Mean hot-on RFP expression shows a sharp increase with temperature above 37°C, consistent with previous work. Meanwhile, the mean cold-on GFP expression response is similar to the standalone TcI39 activation operon, with slight upshifting of the transition temperature.

We illustrated the differential expression of RFP and GFP above and below 39 °C using E. coli incubated at 37 and 44°C:

Schematic illustrating experiment to show differential gene expression with temperature on solid culture. Created with BioRender.
Illustrations drawn with bacteria on agar plates. A plate grown at 37 Celsius shows a green rosebud and a plate grown at 44 Celsius shows a magenta rose in bloom.

Transcriptional activation by TcI mutants represents a cool new tool for global and local thermal control of cells.

Data tools

All flow cytometry data analysis and visualization performed in Python.

Analysis Visualization
Cytoflow Matplotlib
Pandas Bokeh
SciPy HoloViews
Colorcet
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View data analysis code on GitHub

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