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Use of Protein Stability to Develop Dual Luciferase Toxicity Bioreporter Strains
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  • Use of Protein Stability to Develop Dual Luciferase Toxicity Bioreporter Strains
  • Use of Protein Stability to Develop Dual Luciferase Toxicity Bioreporter Strains
저자명
Mitchell. Robert J.,Gu. Man-Bock
간행물명
Biotechnology and bioprocess engineering
권/호정보
2011년|16권 6호|pp.1254-1261 (8 pages)
발행정보
한국생물공학회
파일정보
정기간행물|ENG|
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이 논문은 한국과학기술정보연구원과 논문 연계를 통해 무료로 제공되는 원문입니다.
서지반출

기타언어초록

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 $42^{circ}C$, the FF-Luc activity was assayed. This was possible because a shift from 28 to $42^{circ}C$ for 10 min was sufficient to denature/inactivate the Lux proteins to background levels. After treatment, the substrate for FFLuc 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.