Department of Civil, Environmental, and Architectural Engineering, University of Colorado at Boulder, 1111 Engineering Dr., UCB Box 428, Boulder, CO 80309, USA.
Appl Environ Microbiol. 2011 May;77(9):2863-8. doi: 10.1128/AEM.01915-10. Epub 2011 Mar 4.
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.
为了满足原位测温的需求，我们对温度敏感载体进行了适应性改造，以便实时报告大肠杆菌细胞内热含量的变化。该模型系统利用载体表达β-半乳糖苷酶的量逐渐增加，以响应通过生物相关范围（22 至 45°C）的逐步温度升高。通过校准的荧光和比色报告器判断，整个大肠杆菌细胞和裂解物均表达出β-半乳糖苷酶活性的显著可重复变化，对小于 1°C（35 至 45°C）的温度变化敏感。该模型系统表明，细胞热含量的变化可以独立于细胞维持的介质来检测，这在介质不均匀或非水相，或者介质的传热能力较低的情况下尤为重要。我们在此报告，使用可靠的荧光报告系统，从细胞尺度上可以可靠地实时获得细胞内温度，检测范围为 20°C，在 35 至 45°C 之间的灵敏度至少为 0.7°C。
