KR20030088315A - Bacterial biosensor bearing gene fusion constructed with dnaK promoter of Psudomonas sp. DJ-12, and detection method of aromatic pollutants with the same - Google Patents

Abstract

PURPOSE: A bacterial biosensor using dnaK promoter of Pseudomonas sp. DJ-12 and a detection method of aromatic pollutants using the same biosensor are provided. The biosensor has improved luminescence intensity and sensitivity. CONSTITUTION: A genetic construct for a biosensor comprising dnaK promoter of Pseudomonas sp. DJ-12(KCTC 8967P) and a luciferase gene operably linked to the dnaK promoter is provided, wherein the dnaK promoter of Pseudomonas sp. DJ-12(KCTC 8967P) is polynucleotide amplified by PCR using a pair of primers having the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2; the dnaK promoter has the nucleotide sequence of SEQ ID NO: 3; the luciferase gene is luxCDABE or luc; the luxCDABE is isolated from Vibrio, Xenorhabdus, Photrhabdus and Photobacterium; the biosensor detects at least one aromatic compound selected from biphenyl, 4-chlorobiphenyl(4CB), 4-hydroxybenzoate(4-HBA) and catechol; the biosensor comprises a cell transformed with the genetic construct; and the cell is E. coli. A detection method of aromatic pollutants comprises exposing the biosensor to aromatic compounds to induce the expression of luciferase and measuring the luminescence intensity by luciferase.

Description

Bacterial biosensor bearing gene fusion constructed with dnaK promoter of Psudomonas sp. DJ-12, and detection method of aromatic pollutants with the same}

The present invention provides a gene construct comprising a dnaK promoter of the Pseudomonas strain, a biosensor comprising the cells transformed with the gene construct, and more specifically, the dnaK promoter and reporter gene of Pseudomonas strain DJ-12, lux CDABE. The present invention relates to a microbial biosensor comprising a genetic structure of and an aromatic compound using the same.

Biosensors have been widely used to detect chemical pollutants in complex environments. Environmental biosensors are part of the whole-cell biosensor, which have several types of stress promoters, bioluminescence and green fluorescence that can specifically react to the toxicity of environmental pollutants. Fluorescence is an advanced bio-monitoring technology that can be easily applied to rivers / lakes, water and sewage sources, soil, and the atmosphere by using genetically modified cells composed of reporter systems.

The response of organisms due to toxic substances prepares for response at the gene level. When contacted with toxic substances, the expression of elemental genes to recover or respond according to the degree of damage is called a stress promoter. These stress promoters have specific responses to specific stresses caused by toxic substances, and the development of genetic recombination technology induces cell-specific stress through the combination of these stress promoters and luminescent genes ( lux , luc ) / fluorescence (GFP) genes. It is possible to manufacture a recombinant luminescent / fluorescent cell line capable of detecting toxic substances. Genetically recombined plasmids by combining stress promoters such as E. coli or Bacillus, Pseudomonas, Salmonella, etc. Genetically modified luminescent microorganisms are made that can induce a change in the amount of luminescence for.

Aromatic compounds, in particular chlorinated aromatic compounds, are serious environmental pollutants due to their lipophilic, persistence and toxicity. Although the use of such aromatic compounds is severely restricted in many countries, distribution in nature is still one of the major causes of environmental pollution. Some biosensors can detect and quantify various compounds such as BTEX compounds, naphthalene, biphenyl chloride, and mercury. These biosensors include nahG (Heitzer, A, et al., Appl. Environ. Microbiol. Vol 60, pp. 1487-1494, 1994), which encodes salicylation hydroxylase, bphA, which encodes biphenyl deoxygenase. (Lyaton, AC, et al., Appl. Environ. Microbiol. Vol. 64, pp. 5023-5026, 1998), and todR that degrades toluene (Applegate, BM, et al., Appl. Environ. Microbiol.vol 64, pp. 2730-2735, 1998).

Tauriainen et al. (Tauriainen, SM et al, Appl. Environ.Microbiol.vol. 63, pp.4456-4461, 1997; Tauriainen, SM et al., Anal.Biochem.vol.272, pp.191-198, 1999) A biosensor using luc gene as a marker gene for measuring toxic heavy metals such as arsenite, antimonite and cadmium was prepared. The luc gene was also used to prepare biosensors made of constructs with the Pu promoter of xylR obtained from the TOL plasmid of Pseudomonas putida mt-2 (Willardson, BM, Appl. Environ. Microbiol. Vol. 64, pp. 1006-1012, 1998). The biosensor investigated the luminescence of BTEX and toluene-like molecules, but the luminescence was not very high.

Cha et al. (Cha, HJ, Appl. Environ. Microbiol. Vol 65, pp. 409-41, 1999). Luminescence by the dnaK :: gfpuv construct using the E. coli dnaK promoter was measured at 2-4% ethanol concentration. In addition, it is reported that after 4 hours of exposure, the luminous intensity is weak.

Van Dyk et al. (Van Dyk, TK, Appl. Environ. Microbiol. Vol. 60, pp. 4124-4127, 1994) and Belkin et al. (Belkin, S., et al., Appl. Envrion. Microbiol. Vol. 62, pp.2252-2256, 1996) produced biosensors by fusion with lux CDABE, a luminescent gene, and promoters of stress shock genes including dnaK, grpE, uspA and katG of E. coli. The biosensor was used to monitor various types of toxic substances such as ethanol, phenol, and heavy metals. Among the biosensors, gene constructs containing dnaK promoters have been reported to have greater sensitivity to toxicants than gene constructs containing other promoters. However, biosensors with the structure of the dnaK promoter and lux CDABE have been shown to respond better to ethanol than aromatic contaminants such as pentachlorophenol, 2-nitrophenol and phenol (Van Dyk, TK, et al., Appl. Environ.Microbiol.vol. 61, pp.1414-1420, 1994). Moreover, it has been found that luminescence of the structure is inhibited by phenol at a concentration of 4,170 ppm or more. Thus, there is still a need for biosensors that can detect various aromatic pollutants more sensitively.

In order to solve the problems of the prior art as described above, an object of the present invention is to provide a gene structure for a biosensor comprising a gene encoding the dnaK promoter and luciferase of the Pseudomonas strain.

Still another object of the present invention is to provide a biosensor transformed with the genetic construct having a high sensitivity and high luminescence to aromatic environmental pollutants, and a method of manufacturing the same.

Another object of the present invention to provide a method for detecting aromatic environmental pollutants using the biosensor.

Figure 1 is a schematic diagram showing the preparation of the dnaK promoter using a PCR reaction using primers and DNA designed from E. coli W3101 and Pseudomonas strain DJ-12.

Figure 2a is a gene construct comprising the dnaK promoter and lux CDABE reporter gene of Pseudomonas strain DJ-12, Figure 2b is a gene construct comprising the dnaK promoter of Pseudomonas strain DJ-12 and the luc reporter gene of firefly 2B is a luminescent gene construct comprising a dnaK promoter of E. coli and a lux CDABE reporter gene.

Figures 3a to 3c is shown by biosensor dnaKp-EC :: lux CDEAB (3a), dnaKp-DJ :: lux CDEAB (3b), dnaKp-DJ :: luc (3c) when exposed to various concentrations of ethanol for 180 minutes It is a graph showing luminescence.

Figure 4 shows the sequence of the dnaK promoter of Pseudomonas strain DJ-12 and E. coli (GeneBank accession No. M10420), and an asterisk (*) shows the sequence differences between the two strains.

5A to 5D are graphs showing the degree of luminescence by a biosensor comprising danKp-DJ :: lux CDABE structure when exposed to biphenyl (5a), 4CB (5b), 4HBA (5c), and catechol (5d). .

6A-6C are graphs showing the extent of luminescence by the dnaKp-DJ :: luc gene construct upon exposure to biphenyl (6a), 4CB (6b), and 4HBA (6c).

In order to achieve the above object, the present invention is a gene construct for biosensor comprising a gene encoding a luciferase operably linked to the dnaK promoter of Pseudomonas strain DJ-12 (KCTC 8967P) and the dnaK promoter (genetic It is about construct.

The present invention also relates to a biosensor comprising a cell transformed with the gene construct and detecting the aromatic compound by measuring the luminescence by intracellular luciferase when the cell is exposed to the aromatic compound.

The present invention also relates to a method for detecting an aromatic compound comprising exposing the biosensor to an aromatic compound that induces the expression of luciferase and measuring the luminescence by luciferase.

Hereinafter, the present invention will be described in more detail.

In the present invention, the dnaK promoter of Pseudomonas strain DJ-12 (KCTC 8967P) is used as the stress promoter. The dnaK promoter of the Pseudomonas strain DJ-12 is preferably a polynucleotide amplified by PCR with the primer pairs shown in SEQ ID NOs: 1 and 2, and more preferably a polynucleotide having a nucleotide sequence shown in SEQ ID NO: 3. Pseudomonas strain DJ-12 of the present invention has been deposited with the Daejeon Biotechnology Research Institute and was given the accession number KCTC 8967P. Details of the culture conditions and the medium for the strain is described in the Republic of Korea Patent No. 0316975 of the inventors.

In one embodiment of the present invention, the PCR promoter for obtaining the dnaK promoter (dnaKp) is a promoter region of the dnaK operon (Cowing, DW, et al., Proc. Natl. Acad. Sci. USA vol. 82 ((9), pp.2679). 1, SEQ ID NOs: 1 and 2, designed below, are shown below.

Forward promoter 5'-GTTAGCGGATCCAAAAGCACAAAAAAT-3 '

Reverse promoter 5'-AGCAGTGAATTCCATCTAAACGTCTCCA-3 '

The promoter pairs are used to amplify the genomic DNA of Pseudomonas strain DJ-12 which can use biphenyl and 4-chlorobiphenyl as carbon and energy sources by amplification by PCR amplification.

The promoter of the present invention is the dnaK promoter of the Pseudomonas strain DJ-12, which has various aromatics such as biphenyl, 4-chlorobiphenyl (4-CB), 4-hydroxybenzoate (4HBA), benzoate and catechol. The compound has been reported to induce the expression of the dnaK gene of Pseudomonas sp. DJ-12 (King, E., et al., Appl. Environ. Microbiol. Vol. 62 (1), pp. 262-265, 1996 Park, SH, et al., Curr. Microbiol. Vol. 43, pp. 176-181, 2001; Park, SH, et al., J. Microbiol. Vol. 36, pp. 93-98, 1998a). Various aromatic contaminants are good for the production of stress shock proteins such as Pseudomonas strain DJ-12 and DnaK of E. coli (Blom, A. et al., Appl.Environ.Microbiol.vol58, pp.331-334). It is reported to act as an inducer.

Conventionally, the dnaK gene of E. coli was used as a stress promoter for a biosensor, and the nucleic acid sequence of the dnaK promoter of Pseudomonas strain DJ-12 and E. coli (GeneBank accession No.M10420) of the present invention is shown in FIG. It is shown in SEQ ID NO: 3 and 4, the asterisk (*) in Figure 4 shows the sequence difference between the two strains.

Compared to the dnaK :: gfpuv structure using the E. coli dnaK promoter of Cha et al., The light emission by the dnaKp-DJ :: lux CDABE structure according to the present invention was much stronger, and the dnaKp- using the E.coli dnaK promoter of Van Dyk et al. The luminescence by dnaKp-DJ :: lux CDABE produced by the dnaK promoter of Pseudomonas strain DJ-12 was much stronger than the EC :: lux CDABE construct. This difference in luminescence is believed to be due to differences in promoter sequences. The sequence of Pseudomonas genus DJ-12 and E. coli dnaK promoter (GeneBank accession No .: M10420) is 6 base pairs different, which is shown in FIG. The difference in nucleotide sequences may result in a difference in the luminescence response to ethanol between the dnaKp-DJ :: lux CDABE and dnaKp-EC :: lux CDABE gene constructs.

Van Dyk et al. Reported that the promoters of the dnaK and grpE stress-shock genes and the constructs of the lux CDABE reporter gene can be used to detect individual chemical toxins such as phenol, 2-nitrophenol, copper, sulfate, and ethanol. However, the dnaKp-DJ :: lux CDABE construct according to the present invention has been shown to respond much more strongly to aromatic compounds than constructs using catabolic genes (eg bphA gene) or stress-shock genes of E. coli strains. .

In the present invention, the gene encoding the luciferase operably linked with the dnaK promoter of Pseudomonas strain DJ-12 includes genes encoding all luminescent genes known as luciferase. For example, it may include lux CDABE encoding luciferase of luminescent microorganisms or a portion of luciferase thereof (luxAB, in this case externally adding an aldehyde substrate to measure activity), and the luc gene encoding eukaryotes luciferase. Can be. The Lux gene can be obtained from the lux operon of all luminescent microorganisms, most of which include Vibrio, Xenorhabdus, Photorhabdus, Photobacterium, and the like. .

As a substance detectable using the biosensor gene construct according to the present invention, aromatic compounds such as biphenyl, 4-chlorobiphenyl (4CB), 4-hydroxybenzoate (4-HBA), and catechol It may include.

The present invention also relates to a biosensor comprising a cell transformed with the gene construct and detecting the aromatic compound by measuring the luminescence by intracellular luciferase when the cell is exposed to the aromatic compound. The cells may be bacteria, yeast, plant cells, or animal cells, preferably bacteria, more preferably E. coli.

In a preferred embodiment of the present invention, a plasmid comprising a reporter gene without a promoter by amplifying the genomic DNA of Pseudomonas strain DJ-12 by PCR amplification using the promoter pair, and digesting the obtained PCR product with a restriction enzyme, For example, the plasmid dnaKp-DJ :: lux CDABE (FIG. 2A) was prepared by introducing into a pUCD615 plasmid. The plasmid is transformed into a microorganism to prepare a microbial biosensor. Alternatively, the dnaK promoter may be linked to a reporter gene to prepare a gene construct, introduced into a plasmid, and transformed into a microorganism. The transforming microorganism is preferably E. coli. Therefore, both the plasmid preparation known to those of ordinary skill in the art and the conventional method of transforming the same into microorganisms can be applied to prepare the microbial biosensor of the present invention.

The present invention also provides a method of detecting an aromatic compound comprising exposing the biosensor to an aromatic compound that induces the expression of luciferase and measuring the luminescence by luciferase. Specific methods for detecting aromatic compounds using the biosensor according to the present invention may be performed by those skilled in the art as appropriately selected. For example, when using microorganisms transformed with dnaKp-DJ :: lux CDABE plasmid, contaminants can be measured using a device for contacting the biosensor cells and a device for measuring the light output of the cells. All known optical measuring devices can be used in the detection method of the present invention, for example photomultipliers, charge coupled devices, luminometers, photometers, optical fiber cables Or a liquid scintillation counter.

The aromatic compounds to be analyzed can be measured using biosensors when adsorbed on the surface, dissolved in a liquid medium, or present in the gas phase. Biosensors can be used to detect contaminants attached to particles, dissolved in aqueous samples, or dispersed in the gas phase. In general, however, it is preferable to measure in the liquid phase. It is preferable that sufficient moisture be used so that the biosensor including the gene construct can function reliably, and the sample is diluted with an appropriate buffer composition before the analysis and cultured in this solution. In addition, the analyte may be separated from the sample before analysis using a selective permeable membrane or a solvent. For example, the gas sample may be passed through a liquid or an appropriate solid adsorbent may be used to concentrate the analyte prior to analysis.

Microbial biosensor comprising the dnaK promoter and luxCDEAB of the Pseudomonas genus strain DJ-12 according to the present invention, and a microorganism bios comprising a gene (eg, dnaKp-EC :: lux CDABE) in which a conventional E. coli dnaK promoter and a lux gene are fused. Comparing the sensors, the dnaKp-DJ :: lux CDABE bioluminescent construct showed a response that is about five times stronger than ethanol compared to dnaKp-EC :: lux CDABE. Biosensors comprising the dnaKp-DJ :: lux CDABE structure according to the present invention are directed to aromatic compounds such as biphenyl, 4-chlorobiphenyl (4CB), 4-hydroxybenzoate (4-HBA) and catechol. Upon exposure, bioluminescence by the dnaKp-DJ :: lux CDABE construct was most sensitive at 80 min exposure to 1 mM biphenyl and 4CB, and the luminescence was also very strong for other aromatic compounds, including the E. coli dnaK promoter. It exhibits much stronger luminescence than biosensors. Thus, biosensors comprising dnaKp-DJ according to the present invention are believed to be most useful for detecting aromatic and other contaminants. Therefore, the present invention can be used to develop a microbial biosensor for detecting aromatic contaminants in water by fusing the dnaK promoter with lux CDABE or luc gene using the dnaK gene of Pseudomonas strain DJ-12.

The present invention will be described in more detail with reference to the following examples, but the following examples merely illustrate the present invention, and the protection scope of the present invention is not intended to be limited to the following examples.

Example

Example 1: dnaKp-DJ :: lux Biosensor Manufacturing Including CDABE

The restriction enzyme, DNA modification enzyme, and T4 DNA ligase used in this example were purchased from POSCO Corporation (Seoul, Korea).

The PCR promoter for obtaining the dnaK promoter (dnaKp) is based on the promoter region of the dnaK operon (Cowing, DW, et al., Proc. Natl. Acad. Sci. USA vol. 82 ((9), pp.2679-2683). It was designed, and is shown in Figure 1 and below.

Forward promoter 5'-GTTAGCGGATCCAAAAGCACAAAAAAT-3 '

Reverse promoter 5'-AGCAGTGAATTCCATCTAAACGTCTCCA-3 '

Pseudomonas strain DJ-12 (KCTC 8967P) was used in which biphenyl and 4-chlorobiphenyl were available as carbon and energy sources. Genomic DNA of Pseudomonas strain DJ-12 was amplified by PCR amplification using the promoter pair. The dnaK gene of Pseudomonas strain DJ-12 obtained by cleaving the obtained PCR product with Bam H1 and EcoR1 was linked to pUCD615 cleaved with Bam H1 and EcoR1 (18). The pUCD615 plasmid contained lux CDABE operon of V.fischeri without promoter to prepare dnaKp-DJ :: lux CDABE (FIG. 2A), a luminescent gene construct.

In order to measure the luminescence of the gene construct, the gene construct was transformed into E. coli XL1-Blue.

Example 2: Strain Preparation Including dnaKp-DJ :: luc Structure

The genomic DNA of Pseudomonas strain DJ-12 was amplified by PCR amplification using the primer pair designed in Example 1. According to the (19) method described in SambrooK et al., The PCR product was introduced into the pBluescript SK (+ /-) vector (stratazine, Lazola, California, USA). The dnaK promoter region cleaved with SacI and XhoI was inserted into a pGL3-based vector carrying a luc gene (Promega Co., Madison, USA) to prepare a dnaKp-DJ :: luc gene construct (FIG. 2C).

In order to measure the luminescence of the gene construct, the gene construct was transformed into E. coli XL1-Blue.

Comparative Example 1: dnaKp-EC :: lux Biosensor Manufacturing Including CDABE

The genomic DNA of E. coli W3101 (8) was amplified by PCR amplification using the primer pair designed in Example 1. The dnaK gene of E. coli W3101 obtained by cleaving the obtained PCR product with Bam H1 and EcoR1 was linked to pUCD615 cleaved with Bam H1 and EcoR1 (18). The pUCD615 plasmid contained lux CDABE operon of V.fischeri without promoter to prepare a luminescent gene construct dnaKp-EC :: lux CDABE (FIG. 2B).

In order to measure the luminescence of the gene construct, the gene construct was transformed into E. coli XL1-Blue.

Example 3 and Comparative Example 2: Measurement of Luminescence upon Exposure to Ethanol

Microbial biosensors with dnaKp-DJ :: lux CDABE prepared from Pseudomonas strain DJ-12 were measured for luminescence upon exposure to various concentrations of ethanol. The experimental results are shown in Figure 3a.

The luminescence of the biosensors prepared in Examples 1 and 2 and dnaKp-EC :: lux CDABE of Comparative Example 1 and dnaKp-DJ :: luc in ethanol concentration was measured in the same manner as in Example. The results are shown in FIGS. 3B and 3C.

3A to 3C, the light generated by the biosensor including the dnaKp-DJ :: lux CDABE of the present invention is generated by the dnaKp-EC :: lux CDABE of Comparative Example 1. Much stronger than That is, when exposed to 3% ethanol for 100 minutes, the luminescence by dnaKp-DJ :: lux CDABE of the present invention was about 5 times brighter than the dnaKp-EC :: lux CDABE structure of Comparative Example 1. The luminescence of the dnaKp-DJ :: luc structure of Example 2 for ethanol was much weaker than the two structures. The luminescence response of the biosensor to ethanol decreased in the order of dnaKp-DJ :: lux DCABE> dnaKp-EC :: lux CDABE> dnaKp-DJ :: luc .

Cha et al. (4) showed that luminescence by the dnaK :: gfpuv construct using the E. coli dnaK promoter was much stronger than luminescence by the dnaKp-DJ :: lux CDABE construct compared to the ethanol measurement. Luminescence by dnaKp-DJ :: lux CDABE made with the dnaK promoter of Pseudomonas strain DJ-12 was much stronger than dnaKp-EC :: lux CDABE made with the E. coli dnaK promoter. This difference in luminescence is believed to be due to differences in promoter sequences. The sequence of Pseudomonas genus DJ-12 and E. coli dnaK promoter (GeneBank accession No .: M10420) is 6 base pairs different, which is shown in FIG. This difference in the nucleotide sequence may lead to the difference in the semi-luminescent response to ethanol between the dnaKp-DJ :: lux CDABE and dnaKp-EC :: lux CDABE structures.

Example 4 Measurement of Luminescence for Aromatic Compounds

According to the method of Tauiainen et al. (21), the degree of emission of the three biosensors obtained in Examples 1 and 2, Comparative Example 2 was measured. All aromatics used in the present invention were purchased from Sigma (St. Louis, USA). A stock solution of 4-hydrobenzoate (4HBA) or catechol (100 mM) was prepared using sterile distilled water, and a stock solution of biphenyl or 4-chlorobiphenyl (100 mM) was prepared using ethanol.

Biosensors containing gene constructs for measuring luminescence were incubated in LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl) at 25 ° C. up to the log phase. The initial pH of the medium was adjusted to pH 7.0 before sterilization.

180 ul of microbial biosensor cultures (106 CFU / ml cells) were transferred to 96-well plates and 0.1-5 mM stock solution was added directly to the wells according to the method of Van Dyk et al. (23). Thereafter, the luminescence was measured using a Berthold luminometer (Autolumat LB935, EG & G Berthold Analytical Instruments, Oak Rige, TN, USA) by incubation at 25 ℃. The same experiment was carried out three times for the aromatic compounds of each concentration, and the value obtained by subtracting the luminescence by the biosensor exposed to the aromatic compound from the luminescence of the untreated biosensor was determined as the relative luminescence unit. In the case of biphenyl and 4CB in ethanol, the luminescence of the biosensor induced by ethanol was used by subtracting the luminescence obtained from the biosensor treated with each aromatic compound. The luminescence reaction of biosensors with dnaKp-DJ :: lux CDABE structures for several aromatic compounds was investigated, and the results are shown in FIGS. 5A to 5D.

Luminescence started when the dnaKp-DJ :: lux CDABE construct according to the invention was exposed to biphenyl, 4CB, and 4HBA 1 mM concentrations. The luminescence was highest when exposed to 1 mM 4CB or 0.2 mM catechol for 90 minutes. The luminescence of the dnaKp-DJ :: lux CDABE constructs against biphenyl, 4CB, and 4HBA was the strongest at 1 mM concentration of each aromatic compound, but no luminescence at higher concentrations.

BphAp :: lux CDABE construct produced by the bph A gene promoter of Ralstonia eutropha ENV307 of Lay et al. (12), and the promoters of the dnaK and grpE stress-shock genes of Van Dyk et al. (23,24) and the lux CDABE reporter Although the structure of the gene has been reported, the dnaKp-DJ :: lux CDABE construct according to the present invention is used in a structure using a catabolic gene (eg, bphA gene) or a stress-shock gene (dnaK and grpE) of an E. coli strain. It has been shown to react much more strongly to aromatic compounds.

Example 5 Measurement of Luminescence for Aromatic Compounds

The luminescence reaction of the aromatic sensor of dnaKp-DJ :: luc in Example 2 was investigated, and the results are shown in FIGS. 6A to 6C. In general, 0.8 mM to 3 mM biphenyl, 4CB, and 4HBA induced luminescence by dnaKp-DJ :: luc biosensor when incubated for 80 to 120 minutes. However, dnaKp-DJ :: luc biosensor dnaKp-DJ :: lux CDABE despite using the same promoter as biosensors and bio that includes dnaKp-DJ :: luc compared to dnaKp-DJ :: lux CDABE biosensor The emission level of the sensor was low.

The present invention provides a microbial biosensor comprising a gene structure of the dnaK promoter of the Pseudomonas strain DJ-12 and a reporter gene, lux CDABE, and a method of using the same, thereby providing much higher luminescence intensity and sensitivity compared to conventional microbial biosensors. It provides a biosensor that is very useful for the detection of contaminants, and has the advantage of detecting contaminants more sensitively and accurately.

Claims (10)

A genetic construct for a biosensor comprising a gene encoding a dnaK promoter of Pseudomonas strain DJ-12 (KCTC 8967P) and a luciferase operably linked to the dnaK promoter. The gene construct according to claim 1, wherein the dnaK promoter of Pseudomonas strain DJ-12 is a polynucleotide amplified by primer pairs shown in SEQ ID NOs: 1 and 2. The gene construct of claim 2, wherein the dnaK promoter is a polynucleotide having a nucleotide sequence shown in SEQ ID NO: 3. The method of claim 1, wherein the luciferase gene isluxCDABE Gene orlucGene constructs that are genes. The gene of claim 4, wherein the lux CDABE is derived from a luminescent microorganism selected from the group consisting of Vibrio, Xenorhabdus, Photrhabdus, and Photobacterium. Structure. The biosensor of claim 1, wherein the biosensor is a biophenyl for detecting at least one aromatic compound selected from the group consisting of biphenyl, 4-chlorobiphenyl (4CB), 4-hydroxybenzoate (4-HBA), and catechol. Gene construct that is a sensor. A biosensor comprising a cell transformed with the gene construct according to any one of claims 1 to 6, wherein the cell detects the aromatic compound by measuring luminescence by intracellular luciferase when the cell is exposed to the aromatic compound. 8. The biosensor of claim 7, wherein the cells are bacteria. The biosensor of claim 8, wherein the bacterium is E. coli. A method for detecting an aromatic compound comprising exposing the biosensor according to claim 7 to an aromatic compound that induces the expression of luciferase and measuring the luminescence by luciferase.