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 ␤-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 ␤-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.Temperature is a master environmental variable, whose accurate measurement is critical for understanding biological processes from the molecular to the biome level (13,15). At a molecular level, temperature in biological systems is typically measured by observing the surrounding growth medium, and while the rapid thermal relaxation of the cellular milieu and aqueous medium makes this approximation valid under many circumstances, there are notable exceptions. Rapid cellular and subcellular heat content changes have been theorized to result from alternating electromagnetic fields (EMF) (7), and the continued development of hyperthermia-based therapies combining nanomaterials and EMF (11) requires the development of in situ and in vivo temperature measurement for subcellular and tissue levels.Cellular heat content affects biomolecular assembly, structure, and function. It has long been recognized that cellular systems sense temperature through various means, including transcription factor binding and mRNA secondary structure (3,5,13,15,16,21). In vivo, these systems maintain homeostasis by responding to environmental temperature changes (5,6,16,(20)(21)(22). Subtle changes in biomolecular structure alter the temperature response, as evidenced by temperature-sensitive mutations (2). While these systems have been engineered or are otherwise exploited for functional studies and expression control, none of these heat-sensing systems have been developed for intracellular thermometry (15). We report here the first demonstration of a biomolecular intracellular thermometer. This system utilizes temperature-sensitive mutants of lacI controlling LacZ expression (3, 4), which accurately reports cellular temperature in an environmentally relevant range.Constitutive expression of LacI(Ts) by a T7 promoter (derived from pET24; Novagen/EMD, Darmstadt, Germany) results in the availability of LacI(Ts) to bind lacO sites. At permis...

Optical readout of the intracellular environment using nanoparticle transducers

“…In contrast, photoacoustic thermometry can provide a precision of about 0.2 °C [15], which is similar to the 0.1 to 0.8 °C precision obtained from fluorescent dyes of various kinds [11,16]. The activity of temperature-sensitive enzymes is another possible probe, and this gives a precision of about 0.7 °C [17]. Unfortunately, the latter two transduction modalities are also sensitive to the pH and ionic strength of the intracellular environment, both of which are variable.…”

Thermometry at the nanoscale

“…McCabe et al 173 reported the first demonstration of a biomolecular intracellular thermometer through temperature-sensitive mutants of lacI controlling LacZ expression which is sensitive to the intracellular heat content of Escherichia coli in near-real time.…”

Mapping Intracellular Temperature Using Green Fluorescent Protein

“…Two alternative approaches have been reported very recently. The first is based on transfecting E.coli bacteria with a temperature sensitive vector which increases quantities of β-galactosidase in response to a temperature increment 9 . The heating of individual bacteria was monitored via an increase of the lacZ expression.…”