Electrochemical evaluation of cellular physiological status under stress in Escherichia coli with the rpoS-lacZ reporter gene |
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Authors: | Funabashi Hisakage Ishikawa Miyuki Mie Masayasu Takahashi Fumio Yanagida Yasuko Aizawa Masuo Kobatake Eiry |
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Affiliation: | Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Tokyo, Japan. |
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Abstract: | We developed an electrochemical detection method for evaluating cellular physiological status based on the stringent response as a means to monitor cell viability. A reporter plasmid was constructed by inserting the beta-galactosidase gene (lacZ) under the control of the rpoS promoter, and then used to transform E. coli cells. Electrochemical responses from the products catalyzed by beta-galactosidase expressed by these E. coli cells were detected using the chronoamperometric technique in a nondestructive manner. Comparisons of response currents between the relA-positive strain and relA-negative strain revealed that increases in these currents were caused by the stringent response due to the stressful alcoholic environment, and thus as a model of stressful cultivating conditions. The current was proportional to the beta-galactosidase activity assayed by a conventional method that required the destruction of cells. The cellular physiological status, which depends on the stringent response as a viability marker, therefore, could then be evaluated online with a current using the rpoS-lacZ reporter gene in the relA-positive strain without pretreatment. |
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Keywords: | bioprocess monitoring cellular physiological status electrochemical monitoring rpoS promoter stringent response |
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