Regular articles

Application of internal standard method in recombinant luminescent bacteria test

Dan Li , Miao He , Yong-zhi Wang


Received November 06, 2014,Revised March 07, 2015, Accepted March 16, 2015, Available online June 26, 2015

Volume 35,2015,Pages 128-134

Mercury and its organic compounds have been of severe concern worldwide due to their damage to the ecosystem and human health. The development of effective and affordable technology to monitor and signal the presence of bioavailable mercury is an urgent need. The Mer gene is a mercury-responsive resistant gene, and a mercury-sensing recombinant luminescent bacterium using the Mer gene was constructed in this study. The mer operon from marine Pseudomonas putida strain SP1 was amplified and fused with prompterless luxCDABE in the pUCD615 plasmid within Escherichia coli cells, resulting in pTHE30–E. coli. The recombinant strain showed high sensitivity and specificity. The detection limit of Hg2 + was 5 nmol/L, and distinct luminescence could be detected in 30 min. Cd2 +, Cu2 +, Zn2 +, Ca2 +, Pb2 +, Mg2 +, Mn2 +, and Al3 + did not interfere with the detection over a range of 10− 5–1 mM. Application of recombinant luminescent bacteria testing in environmental samples has been a controversial issue: especially for metal-sensing recombinant strains, false negatives caused by high cytotoxicity are one of the most important issues when applying recombinant luminescent bacteria in biomonitoring of heavy metals. In this study, by establishing an internal standard approach, the false negative problem was overcome; furthermore, the method can also help to estimate the suspected mercury concentration, which ensures high detection sensitivity of bioavailable Hg2 +.

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