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Mitchell, Robert J.
Applied & Environmental Microbiology Lab (AEML)
Research Interests
  • Pathogens, bdellovibrio bacteriovorus, patho-biotechnology

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Use of protein stability to develop dual luciferase toxicity bioreporter strains

Cited 1 times inthomson ciCited 1 times inthomson ci
Title
Use of protein stability to develop dual luciferase toxicity bioreporter strains
Author
Mitchell, Robert J.Gu, Man Bock
Keywords
After-treatment; Background level; E. coli; Firefly luciferase; Fusion genes; Luciferase; Promoter activities; Protein stability; Relative activities; Reporter gene; Reporter protein; SIMPLE protocol; Single compound
Issue Date
201112
Publisher
KOREAN SOC BIOTECHNOLOGY & BIOENGINEERING
Citation
BIOTECHNOLOGY AND BIOPROCESS ENGINEERING, v.16, no.6, pp.1254 - 1261
Abstract
This study presents a simple protocol to measure 2 promoter activities within a single culture when using both Lux and firefly luciferase (FF-Luc) reporters. To demonstrate this, 2 E. coli strains were constructed using 2 compatible plasmids, one harboring a katG::luc fusion gene and the other either a fabA::lux or grpE::lux fusion gene. To differentiate between the FF-Luc and Lux activities within E. coli, we used the instability of the V. fischeri Lux proteins. Basically, it involved a two step assay where (1) without addition of luciferin, only the Lux activity was assayed and (2) with added luciferin and a heat treatment at 42A degrees C, the FF-Luc activity was assayed. This was possible because a shift from 28 to 42A degrees C for 10 min was sufficient to denature/inactivate the Lux proteins to background levels. After treatment, the substrate for FF-Luc was added and the FF-Luc activity could be reliably measured. Using this protocol, it was possible to assay the activities of both bioluminescent reporter proteins and, thus, the relative activity of the different promoters. Subsequent experiments were performed using known inducers of the katG, fabA and grpE promoters where tests were successfully performed with single compound samples as well as samples causing a variety of stresses. These results clearly demonstrated that two promoter activities can be monitored in a single host with this dual-luciferase system.
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DOI
http://dx.doi.org/10.1007/s12257-011-0184-6
ISSN
1226-8372
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