KR20040067437A - Recombinant bacteria, which detect DNA damage and oxidative damage simultaneously - Google Patents

Abstract

PURPOSE: Recombinant bacteria which detect DNA damage and oxidative damage simultaneously are provided, thereby sensitively detecting DNA damage by increasing stability of plasmid in a cell, and improving confidence of data by inhibiting loss of reporter signal when a microorganism is replicated. CONSTITUTION: A recombinant plasmid pRGDK1 contains recA::GFPuv4 gene and katG::luxCDABE gene simultaneously. A recombinant bacteria E. coli RFM445/pRGDK1(EB-Duo-1)(KCTC 10369BP) is produced by transforming E. coli RFM443 with the recombinant plasmid pRGDK1. A method for preparing a plasmid inducing fluorescence and luminous expression comprises locating a promoter which is induced by various stresses in front of two reporter gene of fluorescence and luminous genes and fusing the genes, wherein two genes are fused to have an opposite transcription direction each other.

Description

Recombinant bacteria, which detect DNA damage and oxidative damage simultaneously}

The present invention relates to a recombinant bacterium capable of simultaneously detecting gene damage and oxidative damage, and more particularly, to construct a recombinant plasmid containing katG :: luxCDABE and recA :: GFPuv4 genes simultaneously and introducing them into E. coli cells. When detecting a substance that induces oxidative damage, the luminescence is increased. When a substance that induces genetic damage of cells is detected, the fluorescence is transformed to increase the fluorescence. The present invention relates to a recombinant bacterium capable of sensitively assessing the degree of toxicity on a cell by dividing it into two species through one strain.

Attempts to detect the toxicity of water samples and chemicals using the luminescence of bacteria have been actively attempted for more than a decade, especially after the identification of the luminescent microorganism Vibrio fisheri in the ocean. Has developed. In particular, the Vibrio Fischer makes use of Microtox from Azur Environment of the United States. Has developed a toxicity detection kit called) and is currently being actively used in many ecotoxicological experiments. Toxicity evaluation method using microorganisms has the advantage of simplicity of experiment method, short reaction time for sample, and easy measurement by simplifying the system because it does not require a large number of individuals. However, by using natural luminescence, it is possible to determine whether the substance to be tested is toxic and its intensity by reducing the light. There is no problem.

Recombinant bacteria for conventional hazard assessment had to use only one reporter gene and promoter to evaluate one type of toxicity and stress per strain. For example, even if different promoters are used using the same reporter gene system, it is difficult to separate the signals using a single strain to evaluate several types of toxicity at the same time. In addition, as the present inventors filed in Korean Patent Application No. 2002-57633, filed on September 23, 2002, a method for producing by simultaneously transforming a plurality of plasmids containing each reporter gene and promoter in one microbial species However, this causes a problem of inhibition of sensitivity due to the different number of plasmid replication individual, and the loss of the signal of the reporter that requires different stability of each plasmid during individual replication of the microorganism.

Therefore, the present inventors have studied to solve the above problems, and as a result, using the luminescence and fluorescent genes as reporter genes at the same time, combining different stress promoters in front of each reporter gene to fuse them in one plasmid and one strain The present invention has been completed by developing a recombinant luminescent bacterium capable of simultaneously detecting at least two or more toxic species.

Accordingly, the present invention provides a recombinant luminescent bacterium E. coli RFM443 / pRGDK1 (EB-Duo-1) [KCTC 10369BP] and its chemicals capable of simultaneously detecting chemical or water oxidatively damaging genes and genetically damaging substances and evaluating their toxicity. Its purpose is to make it.

In addition, the present invention is to provide a method for simultaneously detecting the toxicity and harmfulness of oxidative damage and gene damage using the recombinant bacteria.

1 is a schematic diagram showing the construction of the recombinant plasmid pRGDK1.

Figure 2 is a graph showing the fluorescence change over time after treatment of the gene damaging material mitomycin C to recombinant bacterial E. coli RFM443 / pRGDK1 (EB-Duo-1) [KCTC 10369BP].

Figure 3 is a graph showing the luminescence change over time after treatment with hydrogen peroxide, an oxidative damaging agent, to recombinant bacterial Escherichia coli RFM443 / pRGDK1 (EB-Duo-1) [KCTC 10369BP].

The present invention relates to a recombinant plasmid pRGDK1 comprising both the recA :: GFPuv4 gene and katG :: luxCDABE gene, and transformed into recombinant E. coli RFM443 / pRGDK1 (EB-Duo-1) [KCTC 10369BP] transformed by introducing the plasmid into E. coli RRM443. It is characterized by.

In addition, one species of inducing fluorescence and luminescence expression is characterized by placing a promoter induced by different kinds of stress in front of each of two reporter genes (a luminescent gene, a fluorescence gene) and fusion into one plasmid. The production method of the plasmid is another feature.

In addition, a method for simultaneously detecting the toxicity and harmfulness of gene damage and oxidative damage by the fluorescence and luminescence intensity measurement using the recombinant bacteria is another feature.

Referring to the present invention in more detail as follows.

The present invention is to produce a recombinant plasmid containing the katG :: luxCDABE and recA :: GFPuv4 gene at the same time and introduced into the E. coli to detect the substance causing the oxidative damage of the cell to increase the luminescence, the gene of the cell When detecting a substance that induces damage, it is transformed to increase fluorescence, so that the degree of toxicity of a chemical to cells according to the reporter gene can be classified into two species through a single strain. It relates to recombinant bacteria.

First, the katG promoter of KatG protein whose expression is increased by oxidative damage (PkatG) is combined with the lux operon, a luminescent gene, to detect oxidatively damaging substances. The recA promoter (PrecA) of the overexpressed RecA protein was combined with GFPuv4, a fluorescent gene, to prepare each fusion reporter system, and then the two reporter gene systems were fused into one plasmid to construct a recombinant plasmid pRGDK1. Transformants were prepared by transforming with E. coli RFM443. In particular, in the recombinant plasmid production, unlike the prior art, the Lux gene and the GFP gene are positioned so that the transcription directions are reversed in order to fuse the two reporter gene systems to one plasmid. In addition, the position of the promoter for inducing each transcription is placed in front of the Lux gene or GFP gene so as to be opposite to each other (see Fig. 1).

Colonies expressed from the transformants were selected and the change in fluorescence and luminescence sensitivity was confirmed using gene damaging agents and oxidative damaging agents.

The transformed recombinant bacterium Escherichia coli RFM443 / pRGDK1 (EB-Duo-1) was deposited with the Korea Biotechnology Research Institute Gene Bank on November 8, 2002 and received accession number KCTC 10369BP. The recombinant bacterial Escherichia coli RFM443 / pRGDK1 (EB-Duo-1) [KCTC 10369BP] increases the fluorescence intensity when detecting a genetically damaging substance and increases the luminescence intensity when detecting an oxidatively damaging substance.

Therefore, this strain can be used to predict the possibility of oxidative damage and genetic damage of various chemicals at the same time, and can sensitively detect the toxicity and harmfulness of water samples.

In addition, the present invention increases the plasmid stability in cells by using one plasmid compared to the prior art in which two fusion reporter systems are fused and transformed into one plasmid to transform two plasmids. It is possible to detect each damage sensitively and to prevent the loss of the signal of the reporter, which requires different plasmid stability during individual cloning of microorganisms, thereby increasing the reliability of the data obtained. It is expected to be very useful in the field of toxicity assessment of material and water ecosystems.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1 Construction of Recombinant Plasmids

The katG promoter was amplified by PCR using the following primers from strain DPD2511 (Belkins et al., Applied and Environmental Microbiology, 1996, 62: 2252-2256) having katG :: luxCDABE fused plasmid. The following primers were used to amplify 91 at −137.

5'-primer: 5'-GTCCGGATCCGGACATAATCAAAAAAGC-3 '(SEQ ID NO: 1)

3'-primer: 5'-CTGATGGTACCGTCATTTGCCAGTGGC-3 '(SEQ ID NO: 2)

The PCR product was recombined with a multiple cloning site of pDEW201 [Dupont Co., USA], which was treated with BamHI and KpnI restriction enzyme and also had luxCDABE treated with the same restriction enzyme, and named it pDK1.

The recA promoter was amplified from the plasmid containing recA :: luxCDABE using the PCR method. The following primers were used for amplification up to -184 and +33 parts.

5'-primer: 5'-GCCTCTCGGATCCGTCTGGTTTGC-3 '(SEQ ID NO: 3)

3'-primer: 5'-GCCGGGTGGTACCGGATAGTCAAT-3 '(SEQ ID NO: 4).

This PCR product was treated with BamHI and KpnI restriction enzymes and recombined with the plasmid vector pDML1, which was also treated with the same enzyme, to make pDMLR2. pDML1 is the pDEW201 plasmid [DuPont, Co. U.S.A.] and pMECA plasmids [Thomson J.M. and Parrott W.A. Biotechniques 1998 24: 922-928] were treated with EcoRI and HindIII restriction enzymes, respectively, to combine to form pDM1 and to incorporate the lux operon into the pLITE1 plasmid [Marincs F. and White D.W. Applied and Environmental Microbiology 1994 60: 3862-3863] was prepared in combination with pDM1 by treatment with EcoRI restriction enzyme. The prepared pDMLR2 plasmid was treated with KpnI and PvuII restriction enzymes, and also pGFPgcn4 [Ito, Y et al. Biochem Biophys Res Commun. 1999 264: 556-560 to prepare a pRG7 plasmid. This constituted a fusion gene of recA :: GFPuv4. Finally, the following method is used to configure pACRG43. recA :: GFPuv4 was amplified by PCR in the pRG7 plasmid. As primers for PCR, 5'-primer of SEQ ID NO: 3 and 3'-primer of SEQ ID NO: 5 (5'-CTTCAAGAATTCATTATTTGTAGAGCTCATCC-3 ') were used. The PCR product was put into TA cloning plasmid pGEM-T easy vector [Promega, USA] to prepare pRGTA. EcoRI treatment from pRGTA yielded the recA :: GFPuv4 region. This was also recombined with EcoRI treated pDK1 to place two reporter genes in one plasmid to finally produce katG :: luxCDABE and recA :: GFPuv4 to be transcribed in opposite directions. ].

Example 2: Construction of Recombinant Bacteria

The plasmid pRGDK1 prepared in Example 1 was introduced into E. coli RFM443 and transformed. pRGDK1 plasmid has ampicillin resistance gene and selected colonies containing pRGDK1 in LB medium containing ampicillin, and luminescent / fluorescent expression of selected colonies was induced by mitomycin C, a substance that induces gene damage in cells. Hydrogen peroxide, a substance that induces damage, was identified.

The transformed recombinant bacterium Escherichia coli RFM443 / pRGDK1 (EB-Duo-1) [KCTC 10369BP] was deposited with the Korea Biotechnology Research Institute Gene Bank on November 8, 2002 and was assigned accession number KCTC 10369BP.

Example 3: Confirmation of Genetic Damage and Oxidative Damage Detection of Transformed Recombinant Luminescent E. Coli RFM443 / pRGDK1 (EB-Duo-1) [KCTC 10369BP]

Recombinant bacterial Escherichia coli RFM443 / pRGDK1 (EB-Duo-1) [KCTC 10369BP] was incubated with absorbance of 0.08 (600 nm) in 100 ml LB medium containing 25 μg / ml of ampicillin. To determine the luminescence intensity, 100 μl of the cultured cell solution is taken and the chemical to be tested in each well (concentration of hydrogen peroxide: 0.62, 1.24, 2.47, 4.94, 9.88, 19.75, 37.5, 75, 150 ppm or mitomycin C Concentrations: 0.039, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 ppm) were dispensed into opaque 96 well plates [Microtiter ™, DYNEX technologies, USA], respectively. Likewise, 100 μl of the cultured cell solution was taken to measure the fluorescence intensity and dispensed into a transparent 96 well plate (Tissue culture tesplate, Young Sciences Inc., South Korea) containing the chemical to be tested in each well. Opaque plates are placed in a 96 well luminescence measuring device [Microtiter plate reader, MLX, USA] to measure luminescence intensity, and transparent plates are placed in a 96 well fluorescence measuring device [FL600, BioTek, USA] to measure fluorescence intensity. Put in. After 6 hours, the degree of luminescence and fluorescence intensity were simultaneously measured, and the luminescence or fluorescence changes according to chemical concentrations were simultaneously monitored. FIG. 2 shows the inoculation of mitomycin C, a gene damaging substance, and hydrogen peroxide, a oxidative damaging substance, by concentration, in which recombinant bacterial Escherichia coli RFM443 / pRGDK1 (EB-Duo-1) [KCTC 10369BP] emits a fluorescent signal to detect a gene damaging substance. This is the result of checking whether it can be selectively detected. As shown in (a) of Figure 2 it can be seen that after 4 hours the fluorescence intensity for the concentration of the gene damage material is changed, which is increased in proportion to the concentration. However, in FIG. 2 (b), the fluorescence intensity did not show any difference according to the hydrogen peroxide concentration as compared with the control group. This is because it was engineered to fluoresce against the recombinant bacterial Escherichia coli RFM443 / pRGDK1 (EB-Duo-1) [KCTC 10369BP] gene damage but to increase the degree of luminescence for oxidative damage. FIG. 3 shows the inoculation of mitomycin C, which is a gene damaging substance, and hydrogen peroxide, which is an oxidative damaging substance, by concentration. Recombinant bacteria E. coli RFM443 / pRGDK1 (EB-Duo-1) [KCTC 10369BP] oxidizes the luminescent signal. This is the result of checking whether the damage substance can be selectively detected. As shown in (a) of FIG. 3, it could be seen that the emission intensity with respect to the concentration of hydrogen peroxide varies from the beginning. That is, the luminescence intensity is increased to a certain level of hydrogen peroxide concentration. However, in Fig. 3B, the luminescence intensity did not increase at all. This is because recombinant Bacterial Escherichia coli RFM443 / pRGDK1 (EB-Duo-1) [KCTC 10369BP] was engineered to emit light for oxidative damage but increased fluorescence intensity for gene damage.

As described above, the present invention is to produce a recombinant bacterial Escherichia coli RFM443 / pRGDK1 (EB-Duo-1) [KCTC 10369BP] by fusion of two reporter genes in one plasmid to increase the stability of the plasmid in the cell more sensitive Each damage can be detected, and the stability of each plasmid can be prevented due to the different plasmid stability when cloning microorganisms, and thus the reliability of the data obtained can be further secured. . Therefore, the recombinant bacterium acts as a detector for analyzing the toxicity of various chemicals and water samples and providing a standard for classifying the hazards into gene damage and oxidative damage simultaneously. It can be usefully used in the evaluation field.

<110> KwangJu Institute of Science and Technology <120> Recombinant bacteria, which detect DNA damage and oxidative          damage simultaneously <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid used as 5'-primer of PCR for amplifying katG          promoter <400> 1 gtccggatcc ggacataatc aaaaaagc 28 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Nucleic aicd used as 3'-primer of PCR for amplifying katG          promoter <400> 2 ctgatggtac cgtcatttgc cagtggc 27 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Nucleic aicd used as 5'-primer of PCR for amplifying recA          promoter <400> 3 gcctctcgga tccgtctggt ttgc 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Nucleic aicd used as 3'-primer of PCR for amplifying recA          promoter <400> 4 gccgggtggt accggatagt caat 24 <210> 5 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Nucleic aicd used as 3'-primer of PCR for amplifying recA :: GFPuv4 <400> 5 cttcaagaat tcattatttg tagagctcat cc 32

Claims (5)

Recombinant plasmid pRGDK1 comprising both a recA :: GFPuv4 gene and a katG :: luxCDABE gene. Recombinant bacterial E. coli RFM443 / pRGDK1 (EB-Duo-1) characterized in that transformed by introducing the recombinant plasmid pRGDK1 into E. coli RFM443 [KCTC 10369BP] Fluorescent and characterized in that the two reporter genes (luminescent gene, fluorescent gene) in front of each other by placing a promoter induced by different kinds of stress to produce two reporter fusion system and fused them in one plasmid Method for producing a plasmid of one species that induces luminescence expression. 4. The manufacturing method according to claim 3, wherein the two reporter fusion systems are fused to have opposite transfer directions. A method for simultaneously detecting the toxicity and harmfulness of gene damage and oxidative damage substances by fluorescence and luminescence intensity measurement using recombinant bacterial Escherichia coli RFM443 / pRGDK1 (EB-Duo-1) [KCTC 10369BP].