Abstract
Engineered microorganisms have proven to be a highly effective and robust tool to specifically detect heavy metals in the environment. In this study, a highly specific pigment-based whole-cell biosensor has been investigated for the detection of bioavailable Hg(II) based on an artificial heavy metal resistance operon. The basic working principle of biosensors is based on the violacein biosynthesis under the control of mercury resistance (mer) promoter and mercury resistance regulator (MerR). Engineered biosensor cells have been demonstrated to selectively respond to Hg(II), and the specific response was not influenced by interfering metal ions. The response of violacein could be recognized by the naked eye, and the time required for the maximum response of violacein (5 h) was less than that of enhanced green fluorescence protein (eGFP) (8 h) in the single-signal output constructs. The response of violacein was almost unaffected by the eGFP in a double-promoter controlled dual-signals output construct. However, the response strength of eGFP was significantly decreased in this genetic construct. Exponentially growing violacein-based biosensor detected concentrations as low as 0.39 μM Hg(II) in a colorimetric method, and the linear relationship was observed in the concentration range of 0.78–12.5 μM. Non-growing biosensor cells responded to concentrations as low as 0.006 μM Hg(II) in a colorimetric method and in a Hg(II) containing plate sensitive assay, and the linear relationship was demonstrated in a very narrow concentration range. The developed biosensor was finally validated for the detection of spiked bioavailable Hg(II) in environmental water samples.
Highlights
Mercury is a naturally occurring heavy metal, and the process of industrialization has resulted in its widespread distribution as an ubiquitous environmental toxin[1]
AmpR, T7 promoter, lac operator pET-21a derivative containing the violacien expression cassette inserted as a NdeI/SacI fragment pET-21a derivative containing merR and Pmer divergent promoter region cloned into BglII and XbaI sites pPmer derivative, an artificial hybrid mer operon with transcriptions of mcherry and egfp under the control of independent Pmer divergent promoter region pET-vio derivative containing merR and Pmer divergent promoter cloned as a BglII/XbaI fragment pPmer derivative, an artificial hybrid mer operon with transcriptions of vioABCDE and egfp under the control of independent Pmer divergent promoter region pPmer derivative carrying promoterless egfp cloned into NdeI and HindIII sites always inevitable[11,12]
The merTPCAD gene cluster was substituted with the violacein biosynthetic gene cluster originated from Chromobacterium violaceum, which is under the control of the mer divergent promoter (Fig. 2)
Summary
Mercury is a naturally occurring heavy metal, and the process of industrialization has resulted in its widespread distribution as an ubiquitous environmental toxin[1]. Existing instrumental analysis methods such as atomic absorption spectroscopy, atomic fluorescence spectrometry, and inductively coupled plasma-mass spectrometry are sensitive. They are mainly used to determine the total amount of elemental mercury[3]. Whole-cell biosensors, which can even reproduce independently, have the potential to complement currently used physical and chemical analysis methods, allowing a preliminary detection of bioavailable heavy metal ions to assess the impact of them on the environment by simulating environmental microorganisms[4]. Novel whole-cell biosensors with stable signal outputs that can be detected conveniently and rapidly are always needed to meet the requirements of practical applications
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