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Development of a device that generates a temperature gradient in a microtiter plate for microbial culture

  • Atsushi Shibai
    Correspondence
    Corresponding authors at: Center for Biosystems Dynamics Research (BDR), RIKEN, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan.
    Affiliations
    Center for Biosystems Dynamics Research (BDR), RIKEN, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan
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  • Hazuki Kotani
    Affiliations
    Center for Biosystems Dynamics Research (BDR), RIKEN, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan
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  • Masako Kawada
    Affiliations
    Center for Biosystems Dynamics Research (BDR), RIKEN, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan
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  • Naomi Yokoi
    Affiliations
    Center for Biosystems Dynamics Research (BDR), RIKEN, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan
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  • Chikara Furusawa
    Correspondence
    Corresponding authors at: Center for Biosystems Dynamics Research (BDR), RIKEN, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan.
    Affiliations
    Center for Biosystems Dynamics Research (BDR), RIKEN, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan

    Universal Biology Institute, School of Science, The University of Tokyo, Faculty of Science Bldg.1, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
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Open AccessPublished:July 28, 2022DOI:https://doi.org/10.1016/j.slast.2022.07.004

      Abstract

      Although temperature is a fundamental parameter in biology, testing various temperature conditions simultaneously is often difficult. In the present study, we developed a device for generating a temperature gradient in arrays of wells on a microtiter plate. This device consists of a pair of Peltier elements and temperature sensors placed on both ends of a flat aluminum bar to generate a linear temperature gradient. The device loads a microtiter plate at the center of the aluminum bar and transfers the temperature gradient to the bottom of the wells in the plate. This device successfully maintained a temperature gradient of 38.2 to 43.1°C on the horizontal axis of a 96-well microtiter plate in an incubator at 31°C. Furthermore, using this device, we demonstrated a laboratory evolution experiment of Escherichia coli, which was selected on the basis of its ability to grow at high temperatures. The developed device also facilitates a two-dimensional assay to determine the effects of temperature and drug concentrations on cellular growth.

      Keywords

      1. Introduction

      Temperature is a fundamental parameter of laboratory experiments in biology, as temperature changes affect all chemical reactions in living cells. Bacterial cells can sense their environmental temperatures via sensory functions and alter their behavior or biological activity, such as growth kinetics and swimming behavior, accordingly [
      • Cybulski L.E.
      • Martín M.
      • Mansilla M.C.
      • Fernández A.
      • De Mendoza D.
      Membrane thickness cue for cold sensing in a bacterium.
      ,
      • Kovářová K.
      • Zehnder A.J.B.
      • Egli T.
      Temperature-dependent growth kinetics of Escherichia coli ML 30 in glucose-limited continuous culture.
      ,
      • Nara T.
      • Lee L.
      • Imae Y.
      Thermosensing ability of Trg and Tap chemoreceptors in Escherichia coli.
      ]. Another example is the pUC19 plasmid, a commonly used cloning vector that changes its copy number depending on the growth temperature of the host cells [
      • Lin-Chao S.
      • Chen W.-T.
      • Wong T.-T.
      High copy number of the pUC plasmid results from a Rom/Rop-suppressible point mutation in RNA II.
      ]. Moreover, The temperature preferences of bacterial cells and the effective antibiotic concentrations are dependent on each other [
      • Silva D.
      • Chong P.
      • Fernando D.M.
      • Westmacott G.
      • Kumar A.
      Virulence factors of Acinetobacter baumannii ATCC 17978.
      ,
      • Cruz-Loya M.
      • Tekin E.
      • Kang T.M.
      • Cardona N.
      • Lozano-Huntelman N.
      • Rodriguez-Verdugo A.
      • et al.
      Antibiotics shift the temperature response curve of Escherichia coli growth.
      ]. Therefore, in biological experiments, the arbitrariness of temperature settings and the parallelism of different temperature conditions are preferable.
      However, the generation and maintenance of various temperature conditions in a laboratory are often difficult. As temperature difference can get homogenized via heat radiation, temperature difference inevitably disappears without mechanics and energy to maintain it. Therefore, in general, an incubator simultaneously maintains one temperature condition. This limitation of temperature setting is inconvenient in contrast to the case of other parameters such as substrate addition, where the concentration can be varied from well to well in a microtiter plate, thereby facilitating different high-throughput assays in a single plate.
      In this study, we designed a device that generates a variety of temperatures in an incubator and conducts heat to cell cultures, for example, cultures in wells on a microtiter plate, to enable high-throughput experiments at different temperatures. Takeuchi et al. [
      • Takeuchi K.I.
      • Nakano Y.
      • Kato U.
      • Kaneda M.
      • Aizu M.
      • Awano W.
      • et al.
      Changes in temperature preferences and energy homeostasis in dystroglycan mutants.
      ] developed a device consisting of a flat aluminum bar, with a pair of controllable Peltier elements on both ends of the bar, to study the temperature preference of nematodes. This device generates a linear temperature gradient on a flat bar while nematodes move on this bar, thus facilitating the visualization of the temperature preference of these organisms. On the other hand, there are existing technologies [
      • Mifflin T.E.
      • Felder R.A.
      Development of simple devices for control of temperature above and below ambient on simple pipetting stations.
      ,
      • Riedel T.E.
      • Cox J.C.
      • Ellington A.D.
      Low temperature microplate station.
      ] and commercially available devices wherein a uniformly temperature-controlled metal block is placed under the bottom of a microtiter plate to keep the temperature of the wells constant (for example, Cole-Parmer, cat no. EW-44175, IL). Inspired by these examples, we attempted to develop a device that maintains a temperature gradient on a flat aluminum bar and conducts heat to a microtiter plate placed on the top of the flat bar.
      In the present study, we introduced a device that maintains a temperature gradient in the rows of aligned wells on a microtiter plate in an incubator. This system consisted of a pair of sets of Peltier element units and temperature sensors placed on both ends of a flat aluminum bar. Using this device, we first performed efficient adaptive laboratory evolution of E. coli at high temperatures. In addition, we conducted a two-dimensional assay to determine the effects of temperature and substrate on the growth of E. coli by maintaining two-dimensional gradients of substrate concentration and temperature.

      2. Materials and Methods

      2.1 Temperature gradient device

      The temperature gradient device for the microtiter plate consisted of a flat aluminum bar, Peltier elements, temperature sensors, and other jigs (Fig. 1). The flat aluminum bar consisted of a smaller aluminum plate, which covered the area of the wells on a microtiter plate at its center. The material grade of the aluminum components was 5052. Then, 3D-printed holding jigs were used to place the microtiter plate on a smaller aluminum plate so that the bottoms of the wells touch the aluminum plate. Four Peltier elements (TES1-12739, Thermonamic Electronics Corp., Jiangxi, China) were placed at the four corners of the base aluminum bar (Fig. 1c). Each pair at the same end in the longitudinal direction of the bar was connected and synchronized. The Peltier elements had a heat radiation fin and a fan above them. Two temperature sensors (TSYS01, TE Connectivity, Schaffhausen, Switzerland) were attached to the base aluminum bar in a centrosymmetric manner (Fig. 1b). A microcontroller (Arduino Uno Rev3, https://www.arduino.cc/) with a motor driver unit (MDD10A, Cytron Technologies, Pulau Pinang, Malaysia) controlled the voltage applied to the Peltier elements. The voltages (V1 and V2 shown in Fig. 1c) were feedback-controlled to keep the temperature reading by the sensors stable at the targeted temperatures.
      Fig 1
      Fig. 1Overview of temperature gradient devices. (a, b) The device holds a microtiter plate at the center of a base aluminum plate (dotted plane in b and c) with 3D-printed jigs. The device has a temperature sensor beside two corners of the microtiter plate. Both ends are heat control units and are covered with heat radiation fins and fans. (c) Overview of the elements described above. Four Peltier elements are attached to the four corners of the flat aluminum bar. At the center of the device, there is a slightly thick aluminum plate that touches the bottom of the wells of the microtiter plate to conduct heat.

      2.2 Calibration of the well temperature

      To calibrate the temperature gradient, we measured the temperature of the water in the wells of the microtiter plate using a thermometer at the temperature settings used in the following culture experiments. For example, for the evolution experiments described below, we placed the device in a 31°C incubator and set the target temperatures of the two temperature sensors at 37°C and 47°C. Then, we placed a 96-well microtiter plate (Corning, Cat. no. 3595, NY, USA) with 200 µL water in each well on the device and left it for 1 h. Following this, we measured the water temperatures in 24 of the 96 wells by inserting a thermometer probe (MC1000, Tokai Hit., Co, Ltd., Shizuoka, Japan). The measurements were carried out at 30 min intervals for six wells simultaneously, resulting in four measurements. We then estimated the temperatures of all the 96 wells via linear interpolation of measurements between these 24 wells.

      2.3 Bacterial strains and growth conditions

      We used the E. coli K-12 substrain MDS42 [
      • Pósfai G.
      • Plunkett G.
      • Fehér T.
      • Frisch D.
      • Keil G.M.
      • Umenhoffer K.
      • et al.
      Emergent properties of reduced-genome escherichia coli.
      ] and the E. coli K-12 hypermutable strain MDS42ΔmutS [
      • Shibai A.
      • Takahashi Y.
      • Ishizawa Y.
      • Motooka D.
      • Nakamura S.
      • Ying B.W.
      • et al.
      Mutation accumulation under UV radiation in Escherichia coli.
      ]. Both the strains were acclimated by culturing at 37°C. We used the chemically defined mM63 minimal medium as growth media [
      • Kashiwagi A.
      • Sakurai T.
      • Tsuru S.
      • Ying B.W.
      • Mori K.
      • Yomo T.
      Construction of Escherichia coli gene expression level perturbation collection.
      ].

      2.4 High temperature resistance evolution experiments

      We placed the temperature gradient device on a shaker in an incubator maintained at 31°C and set the target temperatures of the two sensors at 37°C and 47°C. During incubation, the device was shaken at 600 rpm in a 96-well microtiter plate at its center. The medium was poured into the wells on the microtiter plate at 200 µL/well, and the cells were inoculated into the wells so that the initial optical density at a wavelength of 620 nm (OD620) = 10−5. After 23 h of incubation, the microtiter plate was displaced from the device, and a plate reader (FilterMax F3, Molecular Devices, CA) was used to measure its OD620. Then, among the 12 wells in each row on the plate, we selected the well with the highest temperature at which the OD620 value exceeded a threshold value of 0.1. We sampled cells from the selected wells and diluted them with fresh medium in 12 wells in each row on a new plate so that the initial OD620 was 10−5. This cycle of cell transfer was repeated every day for 14 days. Eight replicate lines were maintained under the same conditions. On the last day, the cells were sampled, frozen, and stored as evolved cells.

      2.5 Comparative assay for determining growth temperatures

      The ancestral and evolved cells were inoculated in 5 ml medium in a test tube and precultured at 40°C with shaking at 150 rpm overnight. The cells were then diluted to OD600 = 10−3 with 5 ml of fresh media in several test tubes and cultured at 37°C, 40°C, 41°C, or 42°C with shaking at 150 rpm. After 18 h, the test tubes were immediately cooled to 4°C, and the OD600 of each cell culture was measured with a spectrophotometer. The experiments were performed in triplicate.

      2.6 Two-dimensional growth assays for measuring temperature and drug concentration

      The temperature gradient device was placed at 30°C, and the target temperatures of the two sensors were set to 35°C and 44°C. During incubation, the device was shaken at 600 rpm, holding a 96-well microtiter plate with 200 µl medium per well medium. Then, kanamycin, a commonly used antibiotic, was added to the wells in seven out of the eight rows of aligned wells on the plate, at seven concentrations, corresponding to each row, ranging from 25.5–22.5 µg/ml, with a serial dilution rate of 20.5, respectively. Simultaneously, all wells were inoculated with cells so that the initial OD620 = 10−4. After 18 h of incubation, the OD of the microtiter plate was measured using a plate reader (FilterMax F3, Molecular Devices, CA).

      3. Results

      3.1 Specifications of the device for generating temperature gradients in wells

      First, we checked whether our device, which directly controls the temperature values at two points on the flat aluminum bar, also indirectly maintains a temperature gradient between the wells touching the bar. We quantified the temperatures of 24 wells in a 96-well microtiter plate using a thermometer. On the aluminum bar with a temperature gradient from 37°C to 47°C, the temperatures of the wells were between 38.2°C to 43.1°C (Fig. 2a). Along with temperature gradient in the longitudinal direction, as expected, there was a slight temperature difference in the short direction. In the short direction, the temperature at the center of the plate was high. Therefore, we considered that the edge effect lowered the temperature of the outer wells on the plate. In addition, we examined how the performance differs among individual devices, by replicating the device and calibrating our device in the same way. We confirmed that there was almost no difference in the temperature gradient between the devices (Fig. S1). These results demonstrate that a 10°C temperature gradient on the aluminum bar corresponds to a 5°C gradient in the wells of a microtiter plate. We assumed that the magnitude of this temperature gradient depended on the power supplied to the Peltier elements and the temperature inside the incubator.
      Fig 2
      Fig. 2Experiments demonstrating the adaptive evolution of E. coli against high temperature stress using the developed device. (a) Water temperature of each well of the 96-well microtiter plate on the device. The color corresponds to the temperature on the color bar. A thermometer measured the temperatures of wells denoted by black squares. Linear interpolation determined the temperatures of the remaining wells. (b) A schematic of subculturing manipulation during the evolution experiment. E. coli cells were inoculated and incubated in 12 wells with a temperature gradient. After one day of incubation, the well with the highest temperature among wells with OD > 0.1 was selected, and its cell culture was sampled. Then, the sampled cells were inoculated into 12 wells on a new microtiter plate for the next round. (c, d) Daily transition of the temperatures of wells for 14 days. The temperature on the y-axis corresponds to the positions of the selected wells in b and the corresponding temperature in a. The experiments were performed in eight replicates. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

      3.2 Continuous increase in the temperatures of culture wells for the 14-day evolution of E. coli

      The temperature-gradient device developed in this study can provide culture conditions at various temperatures. This is a significant advantage for performing selection and screening experiments under high-temperature conditions. To demonstrate this advantage, we conducted adaptive evolution experiments using E. coli under high temperature stress. We repeatedly subcultured cells from a well, with the highest temperature among the wells with sufficient cell growth after a 1-day incubation, out of 12 aligning wells in a row on a temperature gradient, so that the cells were always exposed to the highest temperature they could be resistant (Fig. 2b). After 14 days, we observed a substantial increase in the temperatures with the growth of E. coli MDS42 wild type (WT) and its hypermutator (ΔmutS) in the wells (Fig. 2c, d). We statistically confirmed these increases by comparing the temperatures at the end (day 14) and beginning (day 1) of the experiments (Wilcoxon's signed-rank test, p<0.05). ΔmutS reached a significantly higher temperature than WT at the end. Notably, WT initially showed slightly but significantly higher temperatures than ΔmutS (Mann–Whitney U test, p<0.05). These results suggest that the developed device facilitates efficient adaptive evolution for temperature resistance during laboratory experiments.
      To evaluate the change in high-temperature resistance of the evolved strains, we cultured the evolved and ancestor strains under the same conditions, that is, 37, 40, 41, and 42°C for 18 h. Results revealed that all the evolved lineages of WT showed growth at 41°C, although their ancestors could not survive this temperature (Fig. 3a). Moreover, 4 of the 8 evolved lineages could grow at 42°C. For ΔmutS, all the evolved lineages grew at 42°C; on the other hand, their ancestor could up to 41°C (Fig. 3b). From these results, we confirmed that the obtained cells that evolved during the laboratory evolution experiment, as assessed using our temperature gradient device, could grow at higher temperatures than those required for their ancestors.
      Fig 3
      Fig. 3High temperature-resistant cells were evolutionarily obtained with the device. (a, b) For both WT and ΔmutS, the growth deficient at higher temperatures relative to 37°C, which the ancestral cells usually habitat, was compared between the ancestral lineage (solid line) and the evolved lineages (dashed line). The experiment was replicated three times, and the error bars on the y-axis represent the standard deviations.

      3.3 The device facilitated a two-dimensional cell growth assay between temperature and drug concentration

      Our device generates and maintains a temperature gradient in the longitudinal direction of a microtiter plate, with wells arranged in a two-dimensional plane. Therefore, we thought that combining other parameters, such as drug concentration gradients, in a redundant short direction would realize an experiment that assays the interactions between temperature and drug simultaneously. As an experimental demonstration, we designed 7 of 8 rows of 96-well microtiter plates as a concentration gradient of the antibiotic kanamycin and the remaining 1 row as a drug-free control. We then inoculated E. coli cells into the plate and incubated them on the developed temperature-gradient device. We observed relationships between cell growth and the following factors: the temperature gradient and drug concentration on the horizontal and vertical axis, respectively (Fig. 4). Then, by inferring the temperatures of the wells from their positions, we demonstrated a two-dimensional plot of the effect of temperature and drug concentration on cell growth. This result indicates that the developed temperature gradient device is widely applicable to high-throughput assays of interactions between temperature and arbitrary substrates on a microtiter plate.
      Fig 4
      Fig. 4Demonstration of two-dimensional assays for temperature and drug concentration. Each bar corresponds to a well of a 96-well microtiter plate. The height of the bar represents the cell density of E. coli, and the color corresponds to the temperature of the color bar. Seven of the eight rows represent the dilution series for kanamycin, and the remaining row represents the drug-free condition. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

      4. Discussion

      In the present study, we constructed a device to generate a temperature gradient among wells on a microtiter plate. This device controls a linear temperature gradient between two points on an aluminum bar and conducts heat to the wells from the bottom of the microtiter plate. Using E. coli, we demonstrated that this device achieves efficient high-temperature resistance evolution (Figs. 2 and 3). Moreover, the device facilitated a two-dimensional assay with antibiotic concentration as a parameter, in addition to the temperature gradient (Fig. 4). As it would be easy and inexpensive to manufacture, we expect this device to be widely applicable in biology laboratories, wherein temperature is a fundamental parameter.
      In particular, efficient high temperature-resistant evolution studies using this device can contribute to the development of directed evolution techniques for improving the thermostability of valuable enzymes expressed in microbial cells [
      • Zhang Z.G.
      • Yi Z.L.
      • Pei X.Q.
      • Wu Z.L.
      Improving the thermostability of Geobacillus stearothermophilus xylanase XT6 by directed evolution and site-directed mutagenesis.
      ,
      • Chirumamilla R.R.
      • Muralidhar R.
      • Marchant R.
      • Nigam P.
      Improving the quality of industrially important enzymes by directed evolution.
      ,
      • Peña M.I.
      • Davlieva M.
      • Bennett M.R.
      • Olson J.S.
      • Shamoo Y.
      Evolutionary fates within a microbial population highlight an essential role for protein folding during natural selection.
      ,
      • Blanchard K.
      • Robic S.
      • Matsumura I.
      Transformable facultative thermophile Geobacillus stearothermophilus NUB3621 as a host strain for metabolic engineering.
      ]. Enzymes that can function at high temperatures are preferable in industrial fields because of their rapid kinetics and the advantage of avoiding biological contaminations [
      • Chirumamilla R.R.
      • Muralidhar R.
      • Marchant R.
      • Nigam P.
      Improving the quality of industrially important enzymes by directed evolution.
      ]. The strategy of raising the culture temperature is often a problem in the directed evolution of high temperature-resistant bacteria. When the cells have adapted to one temperature and are then exposed to another higher temperature, an excessive increase in temperature would cause too little growth or extinction, interrupting the directed evolution experiment [
      • Kishimoto T.
      • Iijima L.
      • Tatsumi M.
      • Ono N.
      • Oyake A.
      • Hashimoto T.
      • et al.
      Transition from positive to neutral in mutation fixation along with continuing rising fitness in thermal adaptive evolution.
      ,
      • Rudolph B.
      • Gebendorfer K.M.
      • Buchner J.
      • Winter J.
      Evolution of Escherichia coli for growth at high temperatures.
      ,
      • Kishimoto T.
      • Ying B.W.
      • Tsuru S.
      • Iijima L.
      • Suzuki S.
      • Hashimoto T.
      • et al.
      Molecular clock of neutral mutations in a fitness-increasing evolutionary process.
      ]. The developed device can maintain various temperatures, including the temperature ranges required for the sufficient growth of cells, for the growth of different cell populations simultaneously, avoiding unintended termination of the experiment. In addition, in many cases, the ancestral strains of directed evolution are selected based on their inherent properties such as assimilative capacities [
      • Zhang Z.G.
      • Yi Z.L.
      • Pei X.Q.
      • Wu Z.L.
      Improving the thermostability of Geobacillus stearothermophilus xylanase XT6 by directed evolution and site-directed mutagenesis.
      ,
      • Chirumamilla R.R.
      • Muralidhar R.
      • Marchant R.
      • Nigam P.
      Improving the quality of industrially important enzymes by directed evolution.
      ]. Therefore, two-dimensional experiments with this device would also be helpful for a brief examination of the relationship between the response to culture temperature and other properties of the evolved cells.
      In this paper, we performed pilot experiments using E. coli as a model bacterium in temperature conditions of approximately 37–40°C. However, the available temperature range of the device depends on its ambient temperature (i.e., the temperature setting of the incubator housing it). The temperature range used evokes the application of this device in clinical research. For example, the habitat temperature of most intestinal bacterial flora, including E. coli, is approximately 37°C, which is the typical body temperature for humans [
      • Huus K.E.
      • Ley R.E.
      Blowing hot and cold: body temperature and the microbiome.
      ,
      • Plaza J.J.G.
      • Hulak N.
      • Zhumadilov Z.
      • Akilzhanova A.
      Fever as an important resource for infectious diseases research.
      ]. This device facilitates in vitro verification of the effects of various temperatures on the dynamics of microbial communities, simulating changes in human body temperature. In addition, human body temperature or external temperature affects the infection of pathogenic bacteria or viruses, whereas infectious diseases change the body temperature itself [
      • Plaza J.J.G.
      • Hulak N.
      • Zhumadilov Z.
      • Akilzhanova A.
      Fever as an important resource for infectious diseases research.
      ,
      • Schwab F.
      • Gastmeier P.
      • Meyer E.
      The warmer the weather, the more gram-negative bacteria - impact of temperature on clinical isolates in intensive care units.
      ,
      • Belon L.
      • Skidmore P.
      • Mehra R.
      • Walter E.
      Effect of a fever in viral infections ⇔ the ‘Goldilocks’’ phenomenon?’.
      ]. We expect that this device will enable high-throughput assays of such intricate effects of antibiotics, antivirals, or human antipyretics on pathogens.

      Contributors

      A.S. conceptualized the research; A.S. and C.F. supervised the research project; A.S., H.K., M.K., and N.Y. performed the experiments and the analyses; A.S. visualized the results; and A.S. and C.F. wrote the original draft. All authors reviewed the manuscript.

      Declaration of Competing Interest

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

      Acknowledgments

      This work was funded by the Japan Society for the Promotion of Science KAKENHI grants (grant numbers 17J07299 to A.S. and 19K16114 to A.S.).

      Appendix. Supplementary materials

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