Use of protein stability to develop dual luciferase toxicity bioreporter strains

Robert J. Mitchell, Man Bock Gu

Research output: Contribution to journalArticle

1 Citation (Scopus)

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 42°C, the FF-Luc activity was assayed. This was possible because a shift from 28 to 42°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.

Original languageEnglish
Pages (from-to)1254-1261
Number of pages8
JournalBiotechnology and Bioprocess Engineering
Volume16
Issue number6
DOIs
Publication statusPublished - 2011 Dec 1

Fingerprint

Firefly Luciferases
Protein Stability
Luciferases
Toxicity
Proteins
Escherichia coli
Assays
Fusion reactions
Genes
Gene Fusion
Luminescent Proteins
Heat treatment
Substrates
Plasmids
Hot Temperature
Experiments

Keywords

  • Bioluminescence
  • Luciferase
  • Oxidative stress
  • Promoter fusion
  • Reporter gene

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology
  • Biomedical Engineering
  • Bioengineering

Cite this

Use of protein stability to develop dual luciferase toxicity bioreporter strains. / Mitchell, Robert J.; Gu, Man Bock.

In: Biotechnology and Bioprocess Engineering, Vol. 16, No. 6, 01.12.2011, p. 1254-1261.

Research output: Contribution to journalArticle

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