KR20230155962A - Biosensor for detecting manganese - Google Patents

Abstract

The present invention relates to a microbial-based biosensor for detecting manganese. The bioreporter microorganism of the present invention uses MntR or recombinant MntR as a sensing domain and contains the promoter of the mntS gene linked to the reporter gene as a reporter domain, thereby producing high levels of manganese. It can be detected with target selectivity and specificity, and sensitivity to manganese has been improved by deleting mntP , so it can be used as a biosensor for manganese-specific detection.

Description

Biosensor for detecting manganese {BIOSENSOR FOR DETECTING MANGANESE}

The present invention relates to a microbial-based biosensor for detecting manganese.

Heavy metals are commonly present in the natural world and have a dual effect on living organisms (C.H. Jang, L.L. Cheng, C.W. Olsen, N.L. Abbott, Nano Lett. 6 (2006) 1053.). For humans, trace amounts of many heavy metals are important in maintaining the body's regulatory functions, but when heavy metals accumulate in human organs, they cause various health problems. Since heavy metals do not decompose and are harmful to the ecosystem, monitoring their presence is very important for human health, so sensors for detecting heavy metal ions are important in various fields such as medicine, water quality, air, and food. It is increasing further. Heavy metal ions are known to be a factor in biotoxicity and disease-causing. In particular, heavy metal ions in aqueous solution cause cytotoxicity, and when combined with amyloid fibers in the brain, they are closely related to neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. . Therefore, it is very important to detect target heavy metal ions selectively and at low concentrations.

Currently, measurement methods for monitoring trace amounts of heavy metals include radioactivity analysis using solid samples and alpha (α)-ray-spectrometry, as well as fluorescence analysis using liquid samples, ultraviolet absorptiometry, and ICP (Industively Coupled Plasma) luminescence. These include spectroscopy and ICP mass spectrometry. Recently, with the increase in the development of nano/biosensors, research on the development and use of sensing layers with excellent detection performance is increasing. In particular, research on developing optical biosensors for high sensitivity detection that can utilize selective and specific binding is being conducted. It's increasing

A biosensor is a device consisting of immobilized biological components and a transducer that converts biochemical signals into quantifiable electrical signals. It is an analysis technology that can quickly and accurately detect substances in various environments at low concentrations. Biosensors are largely divided into protein-based and cell-based methods. In terms of biological applicability, selectivity, convenience, and measurement sensitivity, cell-based whole-cell biosensors are used. A lot of research is being done. Whole-cell biosensors use genetically engineered recombinant cells composed of a promoter that can specifically respond to toxicity caused by environmental pollutants and a reporter system such as β-galactosidase or firefly luciferase. It is an advanced bio-monitoring technology that can be applied to measure environmental pollution in rivers, lakes, water and sewage sources, and soil. In the case of conventional biosensors that detect heavy metals, promoters and gene regions that respond to heavy metals are discovered, inserted into a vector, and the transformed host cells are used as biosensors to develop the transformed host cells into biosensors. Research has been conducted.

The purpose of the present invention is to provide a recombinant vector for producing microorganisms for detecting manganese.

Additionally, an object of the present invention is to provide a recombinant microorganism for detecting manganese.

Additionally, an object of the present invention is to provide a biosensor for detecting manganese.

Additionally, an object of the present invention is to provide a method for manufacturing a biosensor for detecting manganese.

Additionally, an object of the present invention is to provide a method for detecting manganese.

Additionally, an object of the present invention is to provide a method for quantifying manganese.

To achieve the above object, the present invention provides a recombinant vector comprising an mntS promoter region and a reporter gene operably linked to the promoter region.

Additionally, the present invention provides a recombinant microorganism transformed with the above recombinant vector.

Additionally, the present invention provides a biosensor for detecting manganese containing the above recombinant microorganism.

Additionally, the present invention provides a method for manufacturing a biosensor for detecting manganese.

Additionally, the present invention provides a method for detecting manganese in a sample.

In addition, the present invention provides a method for quantifying manganese in a sample.

The bioreporter microorganism of the present invention can detect manganese with high target selectivity and specificity by using MntR or recombinant MntR as a sensing domain and containing the promoter of the mntS gene linked to the reporter gene as a reporter domain, and deleting mntP to detect manganese. Since the sensitivity has been improved, it can be used as a biosensor for manganese-specific detection.

Figure 1 is a schematic diagram showing the promoter regions of mntS, mntP, and mntH in the mnt-operon of E. coli (A) and the Mn(II) sensing E. coli-based biosensor (B).
Figure 2 is a diagram confirming the level of reporter gene expression after exposure to Mn(II) of each biosensor produced by transforming the sensing plasmids pMntS-eGFP, pMntH-eGFP, pMntP-eGFP, and pMntP-ribo-eGFPF into E. coli, respectively:
pMntH-eGFP: Biosensor produced by transforming the pMntH-eGFP vector into E. coli;
pMntP-eGFP: Biosensor produced by transforming the pMntP-eGFP vector into E. coli;
pMntP-ribo-eGFPF: Biosensor produced by transforming the pMntP-ribo-eGFPF vector into E. coli; and
pMntS-eGFP: A biosensor produced by transforming the pMntS-eGFP vector into E. coli.
Figure 3 shows the response of the E. coli _pMntS-eGFP biosensor exposed to various concentrations (0.1 to 10 μM) of Mn(II) for various times (0 to 4 hours).
Figure 4 is a diagram confirming the role of MntR using an E. coli cell-based biosensor with a deleted MntR gene including pMntS-eGFP:
BL21 WT mntS GFP: biosensor introducing pMntS-eGFP into wild-type E. coli;
ΔmntR + pMntS-eGFP: biosensor introducing pMntS-eGFP into mntR deletion E. coli; and
ΔmntR + pMntS-eGFP + mntR: Biosensor introducing pMntS-eGFP and recombinant mntR into mntR deletion E. coli.
Figure 5 is a diagram confirming the role of MntR using an E. coli cell-based biosensor with a deleted MntR gene including pMntS-eGFP.
Figure 6 shows an E. coli cell-based manganese-detection biosensor with improved sensitivity produced using E. coli with the mntP gene deleted. This diagram confirms the level of reporter gene expression after exposure to Mn(II).
Figure 7 is a diagram confirming the selectivity for Mn(II) of the E. coli cell-based manganese-detection biosensor.

Hereinafter, the present invention will be described in detail through embodiments of the present invention with reference to the attached drawings. However, the following embodiments are provided as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. , the present invention is not limited thereby. The present invention is capable of various modifications and applications within the description of the claims described below and the scope of equivalents interpreted therefrom.

In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part is said to “include” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.

All technical terms used in the present invention, unless otherwise defined, are used with the same meaning as commonly understood by a person skilled in the art in the field related to the present invention. In addition, preferred methods and samples are described in this specification, but similar or equivalent methods are also included in the scope of the present invention. The contents of all publications incorporated herein by reference are hereby incorporated by reference.

Throughout this specification, the usual one- and three-letter codes for naturally occurring amino acids are used, as well as generally acceptable codes for other amino acids such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), etc. A three-character code is used. Additionally, amino acids referred to by abbreviations in the present invention are described according to the IUPAC-IUB nomenclature as follows:

Alanine: A, Arginine: R, Asparagine: N, Aspartic Acid: D, Cysteine: C, Glutamic Acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine : M, phenylalanine: F, proline: P, serine: S, threonine: T, tryptophan: W, tyrosine: Y and valine: V.

In one aspect, the present invention relates to a recombinant vector comprising the mntS promoter region of the mnt-operon and a reporter gene operably linked to the promoter region.

In one embodiment, the recombinant vector may be used to produce a bioreporter microorganism for manganese detection.

In one embodiment, the mntS promoter region may include the nucleic acid sequence of SEQ ID NO: 1.

In one embodiment, the reporter gene may be a gene encoding an enzyme that catalyzes a color reaction or luminescence reaction of a substrate, an antibiotic resistance gene, or a gene encoding a fluorescent protein, and the enzyme that catalyzes a color reaction of the substrate is horseradish fur. Horseradish peroxidase, alkaline phosphatase, glucosidase, β-galactosidase (LacZ), tyrosinase, and β-glucuronidase (GUS). The enzyme that catalyzes the luminescence reaction of the substrate may be luciferase, and the fluorescent protein may be GFP (Green Fluorescent Protein), eGFP (enhanced green fluorescent protein), YFP (Yellow Fluorescent Protein), or RFP ( It may be any one selected from Red Fluorescent Protein and Aequorin.

In one aspect, the present invention relates to a recombinant microorganism transformed with a recombinant vector.

In one embodiment, the recombinant microorganism may be a bioreporter microorganism for detecting manganese.

In one embodiment, the recombinant microorganism of the present invention may be a microorganism with a deletion of the mntR , mntP , or mntR and mntP genes, and a microorganism with a deletion of the mntP gene is more preferable because the sensitivity for manganese detection increases.

In one embodiment, the recombinant microorganism of the present invention may be further transformed with a recombinant vector containing an mntR gene or a recombinant mntR gene.

In one embodiment, the recombinant mntR gene may encode a MntR variant containing one or more amino acid substitutions selected from the group consisting of V19K, K73L, R77L, and H135A in wild type MntR, and the wild type MntR is SEQ ID NO: 2 and may be encoded with the nucleic acid sequence of SEQ ID NO: 3.

In one embodiment, the microorganism may be Escherichia coli.

In one embodiment, the recombinant microorganism of the present invention may be used to detect heavy metal(loid) ions (As, Cd, Cr, Ni, Hg, Pb, Zn, Au, Sb, Fe, Co and Mn), , It is more preferable that it is for detecting manganese ion Mn(II).

The promoter of the mntR gene of the present invention is the promoter of the Mn(II)-responsive operon, and the promoter of the mntS gene, which is the promoter region of the Mn(II)-responsive operon, has a binding site for MntR, a regulatory protein of the Mn(II)-responsive operon. MntR, a regulatory protein of the Mn(Ⅱ)-responsive operon, represses the transcription of mntH , a Mn(Ⅱ) importer, and activates mntP , an exporter, in the presence of Mn(Ⅱ). (Ⅱ) regulates manganese homeostasis by regulating importer) and MntP. The promoter region refers to a region containing a promoter (regulator binding site), an operator, and genes in between. Since the Mn(II)-responsive operon exists in various bacterial species, the gene sequence of the promoter region is not particularly limited and may be derived from various bacteria having the Mn(II)-responsive operon. Specific examples include Helicobacter pylori, Mycobacterium tuberculosis, Staphylococcus aureus, Escherichia coli, Salmonella enteric, Sequences derived from bacteria such as Acinetobacter baumannii can be used. These may have sequences registered in the NCBI protein database.

The reporter gene is operably linked to the promoter region (corresponding to a structural gene), and when the MntR protein or recombinant MntR protein is separated from the promoter region in the presence of metal ions, especially manganese, it is transcribed and the reporter protein is expressed/produced. The reporter protein is not particularly limited as long as the level of its production can be easily confirmed, and may be, for example, a fluorescent protein.

In the present invention, manganese selectivity was significantly improved by introducing a recombinant MntR protein with a specific amino acid substitution into the biosensor.

In the present invention, the term “microorganism” may be a cell that can be cultured in a liquid medium. The microorganism may be a prokaryotic cell, a eukaryotic cell, or an isolated animal cell that can be cultured in a liquid medium. The microorganism may be, for example, bacteria, mold, or a combination thereof. The bacteria may be gram-positive bacteria, gram-negative bacteria, or combinations thereof. Gram-negative bacteria may be of the genus Escherichia. The gram-positive bacteria may be of the genus Bacillus, Corynebacterium, Lactobacillus, or a combination thereof. The mold may be yeast, Kluveromyces, or a combination thereof. The microorganism of the Escherichia genus may be, for example, Escherichia coli. The E. coli may be DH5α, MG1655, BL21(DE), S17-1, XL1-Blue, BW25113, or a combination thereof.

The present inventors used recombinant vectors pET21a (Bacterial expression vector with T7lac promoter, adds N-terminal T7 epitope tag and C-terminal His tag), pCDFDuet-1 (The vector encodes two multiple cloning sites (MCS) each of which is preceded by a T7 promoter, lac operator, and ribosome binding site (rbs). The vector also carries the pCloDF13 replicon, lacI gene and streptomycin/spectinomycin resistance marker.) was used, but is not limited to this, and expression vectors known in the art are used. It can be used without restrictions. Preferably, recombinant vectors having different antibiotic resistance genes can be used. In such a case, it is easy to confirm that two recombinant vectors have been introduced into the microorganism.

Since the microorganism of the present invention increases the expression of a reporter protein in response to manganese, it can be used to detect manganese, and more preferably, can be used to detect and quantify manganese in a sample.

In one aspect, the present invention relates to a biosensor for detecting manganese comprising the recombinant microorganism of the present invention.

In one embodiment, the detection limit of the biosensor of the present invention may be 0.01 to 10 μM.

In one aspect, the present invention relates to a method for producing a biosensor for detecting manganese, comprising the step of transforming a microorganism with the recombinant vector of the present invention.

In one embodiment, the step of deleting the mntR or mntP gene in the microorganism may be further included before the transformation step.

In one embodiment, the method may further include the step of transformation with a recombinant vector containing the mntR gene or a recombinant mntR gene.

In one embodiment, the recombinant mntR gene may encode an MntR variant comprising one or more amino acid substitutions selected from the group consisting of V19K, K73L, R77L, and H135A in wild-type MntR.

In one aspect, the present invention includes the steps of 1) exposing a test sample to the biosensor of the present invention; and 2) comparing the expression change of the reporter gene in the biosensor of step 1) with the control group.

In one embodiment, the expression level of the reporter gene can be confirmed by the signal intensity of the reporter protein.

In one embodiment, the sample may be a water sample, a soil sample, or a soil leachate sample.

In one aspect, the present invention includes the following steps: 1) confirming the expression of a reporter gene according to the concentration of manganese in the biosensor of the present invention to obtain a quantitative calibration line; 2) exposing the test sample to the biosensor; and 3) substituting the expression level of the reporter gene into the calibration curve.

In one embodiment, the reporter gene may be an egfp gene, and expression of the reporter gene may be expressed as an induction coefficient represented by Equation 1 below:

[Equation 1]

.

In one embodiment, conditions for treating a sample with a bioreporter microorganism may be exposing Mn(II) to a biosensor containing microorganisms at a density of OD 600 of 0.2 to 0.6 for 1 to 4 hours.

The present invention will be described in more detail through the following examples. However, the following examples are only for illustrating the content of the present invention and are not intended to limit the present invention.

Example 1. Production of sensing plasmid

The promoter regions of mntS, mntP, and mntH in the mnt-operon of E. coli are associated with manganese (Mn) homeostasis, and their transcription levels are controlled by endogenous Mn(II). The regions were fused with egfp as a reporter gene to create pMntS-eGFP, pMntH-eGFP, and pMntP-eGFP plasmids. Since it is known that the riboswitch region between PmntP and mntP is involved in the transcription of mntP, The pMntP-ribo-eGFP plasmid (mntP-riboswitch:egfp) was also produced (Table 1). Specifically, the promoter region of the mntS gene (P _mntS ) (SEQ ID NO: 1), the promoter region of the mntP gene (P _mntP ), and the promoter region of the mntH gene (P _mntH ) were extracted from the genomic DNA of E. coli BL21 through polymerase chain reaction. Amplified by polymerase chain reaction and inserted into the BglII and It was named (Figure 1). All constructed plasmids were confirmed by DNA sequencing.

Example 2. E. coli Fabrication of cell-based manganese-detection biosensor

2-1. Fabrication of E. coli-based biosensor and confirmation of manganese sensing characteristics

The sensing plasmids pMntS-eGFP, pMntH-eGFP, pMntP-eGFP, and pMntP-ribo-eGFP prepared in Example 1 were each transformed into E. coli BL21 WT strain to perform E. coli cell-based manganese-detection. Biosensors were manufactured. The fabricated biosensors were cultured overnight and inoculated into new LB (Luria-Bertani) culture medium. After culturing the cells until the optical density at 600 nm (OD ₆₀₀ ) reached 0.3, the cells were exposed to 0 to 100 μM Mn(II), and after 2 hours, the cells were incubated in 50 μM HCl containing 160 mM HCl. After removing LB by resuspending in mM Tris buffer (pH 7.4), the expression level of green fluorescent protein was measured by excitation/emission using a fluorescence spectrophotometer (FluoroMate FS-2, Scinco, Seoul, South Korea) equipped with a 1 nm bandwidth filter. excitation/emission) was measured at a wavelength of 480 nm/520 nm, and the change in fluorescence intensity of each biosensor was converted to an induction coefficient value using Equation 1 below.

As a result, except for the E. coli _pMntS-eGFP biosensor, no significant expression level change was observed after Mn(II) exposure (Figure 2). In the E. coli _pMntH-eGFP biosensor, reporter gene expression was not induced even after exposure to 100 μM Mn(II) (Figure 2A), and in the E. coli _pMntP-eGFP biosensor and E. coli _pMntP-ribo-eGFP biosensor, Expression of the reporter gene was induced regardless of Mn(II) concentration (Figures 2B and C). On the other hand, the E. coli _pMntS-eGFP biosensor showed a decrease in eGFP signal upon Mn(II) exposure (Figure 2D). Additionally, E. coli transformed with promoterless eGFP was tested as a negative control, and no fluorescence was observed (data not shown).

2-2. Confirmation of Mn(Ⅱ) detection ability of biosensor

In order to optimize the conditions for manganese reaction analysis of the biosensor, the E. coli cell-based biosensor E. coli _pMntS-eGFP produced in the above example was exposed to various concentrations (0.1 to 10 μM) of Mn(II) for 0 to 4 hours. After exposure for a period of time, the response to Mn(II) was converted to an induction coefficient value, and its analysis conditions were optimized.

As a result, the fluorescence intensity increased 1 hour after exposure depending on the Mn(II) concentration, and significant changes were observed at 0.5 and 1 μM Mn(II) after 2 hours (Figure 3). Additionally, the Mn(II) biosensor showed the ability to detect 0.05 and 0.1 μM of Mn(II) (Figure 3).

Example 3. Confirmation of MntR role

3-1. Construction of MntR expression plasmid

Genes encoding wild-type MntR and recombinant MntRs (V19K, K73L, R77L, and H135A) in which point mutations were induced by site-directed mutagenesis were amplified from genomic DNA, respectively, and NdeI of pCDF-Duet and insertion into the

3-2. Production of MntR gene deletion E. coli

mntR , an endogenous gene encoding MntR, was deleted in E. coli BL21 using the Quick and Easy E. coli Gene Deletion Kit. Specifically, the FRT (FLP recognition target)-flanked PGK-gb2-neo cassette containing the target gene was amplified and introduced into E. coli BL21(DE3) containing the pRedET plasmid containing the gene encoding the recombinase to generate mntR. The entire ORF (open reading frame) was replaced. Add a 10% arabinose solution to replace the target gene with a kanamycin resistance gene ( kan ), and delete the kan gene in single deletion E. coli to create an mntR -gene-deleted E. coli strain ( E. coli-mntR ). did. Deletion of mntR was confirmed by PCR.

3-3. MntR gene deletion E. coli Production and verification of cell-based biosensors

To confirm whether MntR regulates reporter gene expression under the control of P _mntS , a biosensor was constructed and confirmed using an E. coli strain with the MntR gene deleted as a host cell for the biosensor. Specifically, using the mntR -deleted E. coli produced in Example 3-2 as a host strain, pMntS-eGFP, a sensing plasmid containing egfp as a reporter gene, and pCDF- produced in Example 3-1 were used. The MntR plasmid was transformed into E. coli WT and E. coli - mntR (E. coli with mntR deleted), respectively, to produce E. coli WT_pMntS-eGFP and E. coli mntR_pMntS-eGFP as E. coli cell-based biosensors. and E. coli -mntR_pMntS-eGFP/pCDF-MntR were produced. The biosensor manufactured as in Example 2-1 was exposed to 0-10 μM of Mn(II), the fluorescence of the biosensor was visually confirmed, and the change in the eGFP signal of each biosensor was calculated as the induction coefficient value. Confirmed by conversion.

As a result, E. coli WT ( E. coli WT_pMntS-eGFP) into which pMntS-eGFP was introduced due to the presence of MntR showed a weak green signal under ultraviolet light (Figure 4A), but the green signal was reduced by deletion of MntR. increased significantly (Figure 4B). Additionally, this green signal appeared to decrease again when recombinant MntR was introduced (Figure 4C). Therefore, it was confirmed that transcription of egfp under the mntS promoter was regulated by MntR. In addition, as a result of expressing the eGFP signals of the E. coli WT_pMntS-eGFP and E. coli -mntR_pMntS-eGFP/pCDF-MntR biosensors as induction coefficient values, the biosensors based on wild type (WT) E. coli showed no difference in the eGFP signal. However, the biosensor based on E. coli with MntR deleted did not respond to Mn(II) exposure (Figures 5A and B), which was recovered by introduction of recombinant MntR (Figure 5C). This is consistent with the results shown in Supplementary Figure 1 and is further evidence that MntR plays a role in MntS expression.

Example 4. Improved sensitivity E. coli Fabrication of cell-based manganese-detection biosensor

Although the pMntS-eGFP biosensor showed a Mn(Ⅱ) concentration-dependent signal, the sensitivity to Mn(Ⅱ) was not sufficient, so a biosensor with enhanced (improved) sensitivity to Mn(Ⅱ) was manufactured. To this end, a biosensor for manganese detection with improved sensitivity was manufactured using E. coli with the mntP gene deleted as a host strain. Specifically, to construct E. coli with the mntP gene deleted, the FRT (FLP recognition target)-flanked PGK-gb2-neo cassette was amplified, and E. coli BL21 containing the pRedET plasmid with the gene encoding the recombinase was generated. (DE3) to replace the entire open reading frame (ORF) of mntP and mntR . Add 10% arabinose solution to replace the target genes with the kanamycin resistance gene ( kan ), delete the kan gene in the single-deletion E. coli , and repeat the same procedure in the double-gene-deleted E. coli strain ( E coli-mntR/mntP ) was generated. Deletion of mntR, mntP, and mntR/mntP was confirmed by PCR, and the gene-deleted E. coli strains were named E. coli-mntR , E. coli-mntP , and E. coli-mntR/mntP , respectively, and the sensing plasmid Each biosensor was produced by transfecting each strain with pMntS-eGFP and the pCDF-MntR plasmid prepared in Example 3-1, and exposed to 0 to 5 μM Mn(II) for 2 hours, The induction coefficient value was confirmed.

As a result, the biosensor using the mntP-deleted strain as the host strain showed improved sensitivity to Mn(II) (FIG. 6). The E. coli- based biosensor without mntP deletion showed an induction coefficient value of approximately 2.0 after exposure to 2 and 5 μM Mn(II), whereas the E. coli- based biosensor with mntP deletion showed an approximately two-fold enhancement. showed a signal change (Figure 6). Therefore, since the two strains showed different responses to the same Mn(Ⅱ) concentration, it was inferred that mntP deletion accumulated Mn(Ⅱ) inside the cells, and the sensitivity of the biosensor could be improved through genetic engineering of the host cell. was confirmed.

Example 5. E. coli Confirmation of selectivity of cell-based manganese-detection biosensor

To confirm the selectivity for Mn(II) of the E. coli cell-based manganese-detection biosensor produced in the present invention, E. coli BL21 ( E. coli WT_pMntS-eGFP) containing pMntS-eGFP , E. coli -mntR containing pMntS-eGFP and pCDF-MntR ( E. coli -mntR_pMntS-eGFP/pCDF-MntR), E. coli -mntR/mntP containing pMntS-eGFP and pCDF-MntR ( E. coli-mntR/mntP _ pMntS-eGFP/pCDF-MntR) biosensor with 5 μM of other heavy metal(loid) ions (As, Cd, Cr, Ni, Hg, Pb, Zn, Au, Sb, After exposure to Fe, Co, and Mn) for 2 hours, the induction coefficient value was confirmed.

As a result, all of the above biosensors responded only to Mn(II), and the sensitivity to Mn(II) of the biosensor using the mntP deleted host strain was found to be significantly increased (FIG. 7). Additionally, no concentration-dependent response was observed for other concentrations of heavy metals (data not shown). Therefore, it was confirmed that the E. coli-mntR/ mntP_pMntS-eGFP/pCDF-MntR biosensor is very specific and selective for Mn(II).

Example 6. E. coli Verification of applicability of cell-based manganese-detection biosensor

To evaluate the applicability of the biosensor of the present invention, water and soil samples artificially contaminated with Mn(II) were applied to the biosensor and Mn(II) was quantified. Specifically, artificially contaminated water samples were treated with known concentrations of Mn(II) (1.2, 1.4, 1.6, and 1.8 μM) or mancozeb (manganese/zinc ethylene-bis-dithiocarbamate) (1.0, 10.0, and 50.0 μM). ), and artificially contaminated soil samples were prepared by treating 1g of loamy soil with different amounts of Mancozeb (1.0, 10.0, and 100.0 mg), adding 10mL of distilled water, and incubating at 250°C at 37°C. The leachate extracted by centrifugation for 3 days at rpm was applied to the biosensor. The artificially contaminated soil and water samples prepared in this way were applied to the E. coli cell-based biosensor of the present invention, and the amount of Mn(II) was calculated using a standard curve obtained using 0-5 μM Mn(II). The amount of Mn(II) released from Mancozeb was calculated using a standard curve. To compare the accuracy of biosensors, residual Mn(II) in leachate of soil samples was analyzed using ICP-AES (Inductively coupled plasma atomic emission spectroscopy) of the Korea Institute of Basic Science.

For Mn(II)-injected water samples, when comparing the Mn(II) concentration determined by the biosensor with the original injected concentration, the accuracy of the biosensor test was found to be approximately 95% (Table 2).

Additionally, when water samples contaminated with mancozeb were analyzed with a biosensor, the amount of Mn(II) detected was not proportional to the amount of mancozeb (Table 3), possibly due to the rate of decomposition and purity of the purchased mancozep. It was inferred that

In addition, the amount of Mn(II) detected by the biosensor in leachate isolated from soil samples contaminated with mancozeb increased in proportion to the amount of mancozeb (Table 4). In the case of a soil sample injected with 10 mg of Mancozeb, 12.2 ppm of Mn(Ⅱ) determined using the biosensor was converted to 122 μg of Mn(Ⅱ). This is because 10 mL of distilled water was used for sample extraction, and the result of this analysis was Mancozeb. The reason it does not reflect the total amount of Mn(II) released from Zeb is because some of the Mn(II) is absorbed into the soil.

As confirmed through the above results, the Mn(II) measurement accuracy of the biosensor of the present invention was found to be more than 90%.

Therefore, it was confirmed that mntR is essential in regulating gene expression, and mntP deletion reduces Mn release in E. coli, contributing to improved sensitivity.

Claims (24)

A recombinant vector comprising an mntS promoter region and a reporter gene operably linked to the promoter region. The recombinant vector according to claim 1, wherein the mntS promoter region includes the nucleic acid sequence of SEQ ID NO: 1. The recombinant vector according to claim 1, wherein the reporter gene is a gene encoding an enzyme that catalyzes a color or luminescence reaction of a substrate, an antibiotic resistance gene, or a gene encoding a fluorescent protein. The method of claim 3, wherein the enzymes that catalyze the color reaction of the substrate include horseradish peroxidase, alkaline phosphatase, glucosidase, and beta-galactosidase (β). A recombinant vector selected from -galactosidase, LacZ), tyrosinase, and GUS (β-glucuronidase). The recombinant vector according to claim 3, wherein the enzyme that catalyzes the luminescence reaction of the substrate is luciferase. The method of claim 3, wherein the fluorescent protein is a recombinant protein selected from among GFP (Green Fluorescent Protein), eGFP (enhanced green fluorescent protein), YFP (Yellow Fluorescent Protein), RFP (Red Fluorescent Protein), and Aequorin. vector. A recombinant microorganism transformed with the recombinant vector of claim 1. The recombinant microorganism according to claim 7, wherein the mntR or mntP gene is deleted. The recombinant microorganism according to claim 7, further transformed with a recombinant vector comprising the mntR gene or a recombinant mntR gene. The recombinant microorganism according to claim 9, wherein the recombinant mntR gene encodes a MntR variant comprising at least one amino acid substitution selected from the group consisting of V19K, K73L, R77L, and H135A in wild type MntR. 11. The recombinant microorganism according to claim 10, wherein the wild-type MntR comprises the amino acid sequence of SEQ ID NO: 2. The recombinant microorganism according to claim 7, wherein the microorganism is Escherichia coli. The recombinant microorganism according to claim 7, for detecting manganese (Mn(II)). A biosensor for detecting manganese, comprising the recombinant microorganism of claim 7. A method for producing a biosensor for detecting manganese, comprising the step of transforming the recombinant vector of claim 1 into a microorganism. The method of claim 15, further comprising deleting the mntR or mntP gene in the microorganism before the transformation step. The method of claim 16, further comprising the step of transformation with a recombinant vector containing the mntR gene or a recombinant mntR gene. The method of claim 17, wherein the recombinant mntR gene encodes a MntR variant containing one or more amino acid substitutions selected from the group consisting of V19K, K73L, R77L, and H135A in wild type MntR. Preparation of a biosensor for detecting manganese method. 1) exposing the test sample to the biosensor of item 14; and
2) A method for detecting manganese in a sample comprising comparing the change in expression of the reporter gene in the biosensor of step 1) with the control group. The method for detecting manganese according to claim 19, wherein the expression level of the reporter gene is confirmed by the signal intensity of the reporter protein. 20. The method of claim 19, wherein the sample is a water sample, a soil sample, or a soil leachate sample. 1) Obtaining a quantitative calibration line by confirming the expression of the reporter gene according to the concentration of manganese in the biosensor of item 14;
2) exposing the test sample to the biosensor; and
3) A method for quantifying manganese in a sample comprising substituting the expression level of the reporter gene into the calibration curve. 23. The method of claim 22, wherein the reporter gene is an egfp gene. The method of claim 23, wherein the expression level of the reporter gene is expressed as an induction coefficient expressed by Equation 1 below:
[Equation 1]
.