LacI(Ts)-regulated expression as an in situ intracellular biomolecular thermometer

Abstract

In response to needs for in situ thermometry, a temperature-sensitive vector was adapted to report changes in the intracellular heat content of Escherichia coli in near-real time. This model system utilized vectors expressing increasing quantities of β-galactosidase in response to stepwise temperature increases through a biologically relevant range (22 to 45°C). As judged by calibrated fluorometric and colorimetric reporters, both whole E. coli cells and lysates expressed significant repeatable changes in β-galactosidase activity that were sensitive to temperature changes of less than 1°C (35 to 45°C). This model system suggests that changes in cellular heat content can be detected independently of the medium in which cells are maintained, a feature of particular importance where the medium is heterogeneous or nonaqueous, or otherwise has a low heat transfer capacity. We report here that the intracellular temperature can be reliably obtained in near-real time using reliable fluorescent reporting systems from cellular scales, with a 20°C range of detection and at least 0.7°C sensitivity between 35 and 45°C.

Fig. 1.

Diagram of a LacI(Ts)-controlled thermometric system. At lower temperatures, the constitutively expressed LacI(Ts) can bind to the synthetic lacO sites engineered into the T7A1 promoter, preventing expression of LacZ. As temperatures increase, 2° and 3° structural changes in the LacI(Ts) molecules result in decreased binding at the lacO sites, leading to increasing expression of LacZ. LacZ expression can be detected using colorimetric (X-gal) or fluorometric (C12FDG) substrates.

Fig. 2.

Temperature testing of all four LacI(Ts) vectors from 22 to 45°C with X-gal as the LacZ indicator substrate. Each point represents the average (n = 3) spectrophotometric measurement of the insoluble blue precipitate (OD₆₃₀) at each temperature. Error bars along the x axis represent the temperature variability in the MiniOpticon (0.2°C). Error bars along the y axis represent 1 standard deviation. (Inset) Representative photograph of the LacZ expression responses of the four lacI mutants, showing the increased catalysis of X-gal with increasing temperature.

Fig. 3.

Sensitivity testing of Its265 using X-gal as the indicator substrate. Each point represents the average OD₆₃₀ (n = 15). Significant differences in the OD₆₃₀ between 44.3 and 45°C indicate an assay sensitivity of >0.7°C in this range. The equation represents a polynomial regression on a minimum of eight discrete temperatures (overlaid), with a correlation coefficient of 0.9939. Error bars along the x axis represent the temperature variability in the MiniOpticon (0.2°C). Error bars along the y axis represent 1 standard deviation.

Fig. 4.

Spectrophotometry of the insoluble blue precipitate from a time course of a cell suspension exposed to 40°C, followed by an additional incubation for 20 min at 35°C. Points indicate the average OD₆₃₀ (n = 3); error bars represent 1 standard deviation. Rapid induction of LacZ at 40°C, followed by a plateau at the earliest time points, is observed. Beyond 5 min, a linear increase in LacZ induction can be seen.

Fig. 5.

Fluorescence microscopy of E. coli loaded for 1 h with 20 μM C12FDG in LB medium. (A) Cell suspension maintained at room temperature for 20 min. (B) Cell suspension heated to 40°C for 20 min. (C) A 1:1 mix of heated cells (as in panel B; examples indicated by red arrows) and room temperature cells (as in panel A; examples indicated by yellow arrows).