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Development of a Cell-Free Optical Biosensor for Detection of a Broad Range of Mercury Contaminants in Water: A Plasmid DNA-Based Approach.

ABSTRACT: Mercury (Hg) is one of the main water contaminants worldwide. In this study, we have developed both whole-cell and cell-free biosensors to detect Hg. Genetically modified plasmids containing the merR gene were used to design biosensors. Firefly luciferase (LucFF) and emerald green fluorescent protein (EmGFP) genes were separately introduced as a reporter. Both constructs showed a detection limit of 1 ppb (Hg) in Escherichia coli and the cell-free system. We found that higher concentrations of Hg become detrimental to bacteria. This cytotoxic effect shows an anomalous result in high Hg concentrations. This was also observed in the cell-free system. We found that EmGFP fluorescence was decreased in the cell-free system because of a change in pH and quenching effect by Hg excess. Once the pH was adjusted to 7 and a chelating agent was used, the EmGFP fluorescence was partially restored. These adjustments can only be done in the cell-free system after the GFP expression and not in whole cells where their number has been decreased because of toxicity. Therefore, we suggest the use of the cell-free-system, which not only reduces the total experimental time but also allows us to perform these postexperimental adjustments to achieve higher sensitivity. We would also recommend to perform more measurements at a time with different dilution factors to bring down the Hg concentration within the measurable limits or to use some other chelating agents which can further reduce the excess Hg concentration.

SUBMITTER: Gupta S

PROVIDER: S-EPMC6648214 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Development of a Cell-Free Optical Biosensor for Detection of a Broad Range of Mercury Contaminants in Water: A Plasmid DNA-Based Approach.

Gupta Saurabh S Sarkar Sounik S Katranidis Alexandros A Bhattacharya Jaydeep J

ACS omega 20190530 5

Mercury (Hg) is one of the main water contaminants worldwide. In this study, we have developed both whole-cell and cell-free biosensors to detect Hg. Genetically modified plasmids containing the merR gene were used to design biosensors. Firefly luciferase (LucFF) and emerald green fluorescent protein (EmGFP) genes were separately introduced as a reporter. Both constructs showed a detection limit of 1 ppb (Hg) in <i>Escherichia coli</i> and the cell-free system. We found that higher concentration ...[more]

PMID: 31460039

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Project description:The stable inheritance of the broad-host-range plasmid RK2 is due at least in part to functions within a region located at coordinates 32.8 to 35.9 kb, termed the RK2 par locus. This locus encodes four previously identified genes in two operons (parCBA and parD; M. Gerlitz, O. Hrabak, and H. Schwab, J. Bacteriol. 172:6194-6203, 1990, and R. C. Roberts, R. Burioni, and D. R. Helinski, J. Bacteriol. 172:6204-6216, 1990). The parCBA operon is functional in resolving plasmid multimers to monomers. Analysis of the plasmid stabilization capacity of deletions within this region, however, indicates that this multimer resolution operon is required for stabilization only in certain Escherichia coli strains and under specific growth conditions. The deletion analysis further allowed a redefinition of the minimal functional region as 790 bp in length, consisting of the parD gene (243 bp) and its promoter as well as sequences downstream of parD. This minimal region stabilizes an RK2-derived minireplicon in several different gram-negative bacteria and, at least in E. coli, in a vector-independent manner. By insertional mutagenesis, both the parD gene and downstream (3') regions were found to be required for plasmid stabilization. The downstream DNA sequence contained an open reading frame which was subsequently shown by transcriptional and translational fusions to encode a protein with a predicted size of 11,698 Da, designated ParE. Since the parDE operon requires the presence of the parCBA operon for efficient stabilization under certain growth conditions, the potential role of multimer resolution in plasmid stabilization was tested by substituting the ColE1 cer site for the parCBA operon. While the cer site did function to resolve plasmid multimers, it was not sufficient to restore stabilization activity to the parDE operon under growth conditions that require the parCBA operon for plasmid stability. This suggests that plasmid stabilization by the RK2 par locus relies on a complex mechanism, representing a multifaceted stabilization system of which multimer resolution is a conditionally dispensable component, and that the function(s) encoded by the parDE operon is essential.

Dataset Information

Development of a Cell-Free Optical Biosensor for Detection of a Broad Range of Mercury Contaminants in Water: A Plasmid DNA-Based Approach.

Publications

Development of a Cell-Free Optical Biosensor for Detection of a Broad Range of Mercury Contaminants in Water: A Plasmid DNA-Based Approach.

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