Measurement of firefly luciferase reporter gene activity from cells and lysates using Escherichia coli arsenite and mercury sensors.
The structural gene encoding firefly luciferase from Photinus pyralis is a widely used reporter both in traditional monitoring of gene expression and in bacterial sensors. Its activity can be detected from living cells (in vivo) without disruption or from cell-free lysate (in vitro). We compared the two measurement methods by using an overall toxicity detecting strain Escherichia coli MC1061(pCSS810), a mercury-sensing strain E. coli MC1061(pTOO11), and two new arsenic sensor strains MC1061(pTOO31) and AW3110(pTOO31) which were constructed for this study. Plasmid pTOO31 was constructed by inserting the ars promoter and the arsR gene from plasmid R773 to control firefly luciferase gene expression. Both in vivo and in vitro methods correlated well with the strains tested [correlation coefficients R = 0.99484 and 0.99834] and gave highly comparable results with standard solutions of arsenite or mercury ions and from six environmental water samples spiked with the ions. Use of the in vivo method resulted in lower variation between replicates of the same sample (CVs ranging from 3.9 to 7.2%) and also between different samples (from 8.6 to 25.9%) compared to the in vitro method (CVs ranging from 8.6 to 17.8% for replicates and from 13.1 to 36.3% for different samples).
Department of Biotechnology, University of Turku, Finland.
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S
Tauriainen
M
M
Virta
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W
Chang
M
M
Karp
D000818:Animals; D001149:Arsenates / Q000494:pharmacology*; D003001:Cloning, Molecular / Q000379:methods; D001517:Coleoptera; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D017930:Genes, Reporter; D007700:Kinetics; D008156:Luciferases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008627:Mercuric Chloride / Q000494:pharmacology*; D010957:Plasmids; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism
D001149:Arsenates; D011994:Recombinant Proteins; D008627:Mercuric Chloride; C009277:sodium arsenate; D008156:Luciferases