KR20040026253A - Recombinant bioluminescent/fluorescent bacteria for the detection oxidative damage and DNA damage simultaneously - Google Patents

Abstract

PURPOSE: Recombinant bioluminescent/fluorescent bacteria for detecting oxidative damage and DNA damage simultaneously are provided, thereby detecting at least two kinds of toxic chemicals simultaneously. CONSTITUTION: A recombinant bioluminescent/fluorescent bacterium Escherichia coli RFM443/pACRG43, pDK1 (EBDUAL22) (KCTC 10292BP) is produced by transforming Escherichia coli RFM443 with a recombinant plasmid pACRG43, which is produced by fusion of a gene damage protein RecA-expressing promoter with a fluorescent protein gene GFPuv4, and a recombinant plasmid pDK1, which is produced by fusion of an oxidative damage protein KatG-expressing promoter katG with a bioluminescent gene lux CDABE. A method for detecting oxidative damage and DNA damage simultaneously is characterized by using Escherichia coli RFM443/pACRG43, pDK1 (EBDUAL22) (KCTC 10292BP).

Description

Recombinant bioluminescent / fluorescent bacteria for the detection oxidative damage and DNA damage simultaneously}

The present invention relates to recombinant luminescent / fluorescent bacteria capable of simultaneously detecting gene damage and oxidative damage, and more particularly, recombinant plasmid pDK1 comprising a katG :: luxCDABE gene and a recombinant plasmid comprising a recA :: GFPuv4 gene. pACRG43 was prepared, and these two plasmids were introduced into Escherichia coli to increase the luminescence when detecting a substance causing oxidative damage of the cell, and fluorescent when detecting a substance that induced genetic damage of the cell. It is related to a recombinant bacterium that can be transformed to increase the sensitivity of the chemicals according to the types of reporter genes, and can be sensitively classified into oxidative damage or genetic damage.

Most of the existing luminescent bacteria maintained their constant light in their natural state, and their toxicity was assessed by decreasing the light due to the inhibition of metabolism or the degree of death when exposed to toxic substances [Microbics Corp., Carlsbad, Calif.].

Oxidative damage causes serious damage to microorganisms, plants, and animals that make up the ecosystem by modifying biopolymers such as cell membranes, proteins, and nucleic acids in the form of free radicals, which are free radicals, and degenerative mental disorders such as Alzheimer's disease and diabetes in humans. And it causes fatal diseases that lead to aging and many types of cancer. Therefore, there is an urgent need for a hazard assessment for natural or artificial harmful toxic substances causing such oxidative damage.

To date, the most commonly used method for detecting toxicity using luminescent bacteria is Microtox [Jennings et al., Water Res 2001 Oct; 35 (14): 3448-56], which is evaluated by the degree of death of microorganisms exposed to harmful substances. Therefore, there is a disadvantage in that it cannot exhibit responsiveness to a specific stress such as oxidative damage.

Therefore, the present inventors filed a patent for a recombinant luminescent bacterium capable of detecting oxidative damage in November 2001 (Korean Patent Application No. 2001-71591).

In addition, the Ames method, which is a recognized method for detecting mutants after inoculation of a chemical species to a microbial species, is widely used, but it is not suitable for obtaining fast results due to long test time and complicated test methods. In order to solve this problem, the development of genetic recombinants that can easily detect gene damage substances by combining promoters and gene groups of related genes expressed during gene damage of cells with reporter genes is being accelerated.

On the other hand, the recombinant bacteria for the existing hazard evaluation was forced to evaluate one type of toxicity and stress per strain using only one reporter gene and promoter. 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 view of the above, the present inventors simultaneously used the luminescent and fluorescent reporter genes as reporter genes, and combined different stress promoters before each reporter gene to simultaneously detect at least two or more toxic species with one strain. To develop a recombinant strain that can be. For recombinant strains, E. coli, which has easy culture conditions, was used.The pDK1 plasmid was constructed by combining the katG promoter of KatG protein, whose expression is increased by oxidative damage, with the lux operon, a luminescent gene, to detect oxidative damage. PACRG43 plasmid was prepared by combining the recA promoter of the RecA protein, which is overexpressed by genetic damage, with the fluorescent gene GFPuv4 to detect a gene damaging substance. This plasmid was transformed with E. coli RFM443 at the same time to develop recombinant luminescent / fluorescent bacterium E. coli RFM443 / pACRG43, pDK1 (EBDUAL22) [KCTC 10292BP], which showed increased luminescence by oxidative damage and increased fluorescence by gene damage. The invention has been completed.

Accordingly, the present invention provides a recombinant luminescent / fluorescent bacterium Escherichia coli RFM443 / pACRG43, pDK1 (EBDUAL22) [KCTC 10292BP], which can simultaneously detect chemical or water oxidatively damaging genes and genetically damaging substances and evaluate their toxicity. It aims to do it.

1 is a schematic diagram showing the production of recombinant plasmid pACRG43.

Figure 2 is a schematic diagram showing the production of recombinant plasmid pDK1.

Figure 3 shows the plasmids of the recombinant plasmids pDK1 and pACRG43 by agarose gel electrophoresis [Lane 1: pRG7 / EcoRI, Lane 2: pRGTA / EcoRI-recA :: GFPuv4 PCR confirmation, Lane 3: pACRG43 / EcoRI, Lane 4 : pACRG43 + pDK1 / EcoRI, Lane 5: pDK1 / EcoRI, Lane 6: Lambda HindIII and 100 bp standards marker.

Figure 4 is a graph showing the absorbance change and fluorescence and luminescence change of E. coli EBDUAL22 strain over time after hydrogen peroxide and MNNG inoculation.

The present invention provides a recombinant plasmid pDK1 (katG :: luxCDABE) and pACRG43 (recA :: GFPuv4) and two plasmids simultaneously introduced into Escherichia coli and transformed recombinant luminescent / fluorescent bacterial Escherichia coli, and oxidative damage and gene damage using the same. It is characterized by its toxicity and its detection method.

The present invention will be described in detail as follows.

The present invention relates to a genetically engineered recombinant luminescent bacterium E. coli RFM443 / pACRG43, pDK1 (EBDUAL22) [KCTC 10292BP] capable of evaluating the toxicity of various chemicals. The toxicity and hazards of can be detected simultaneously.

Recombinant bacteria for conventional hazard assessment have the problem of evaluating one type of toxicity and stress per strain using only one reporter gene and promoter, and simultaneously measuring several kinds of stress responses using a single strain. In order to solve this problem, the present inventors introduced a luminescent protein and a fluorescent protein into a reporter gene in one cell to increase luminescence when oxidative damage is detected and increase fluorescence when a gene damage is detected. By developing a recombinant strain that can detect the toxicity of oxidative damage and genetic damage at the same time.

The recombinant strain is a plasmid pACRG43 fused with recA, the expression promoter of RecA protein formed by gene damage, and GFPuv4, the fluorescent protein gene, and katG and the light emitting gene, the expression promoter of KatG protein formed by oxidative damage of cells. It was produced by transforming pDK1, a plasmid fused with luxCDABE.

The colonies expressed from the transformants possessing the recombinant plasmids were selected and the changes in luminescence and fluorescence sensitivity were confirmed using gene damaging agents and oxidative damaging agents. The transformed recombinant luminescent / fluorescent bacterium Escherichia coli RFM443 / pACRG43, pDK1 (EBDUAL22) was deposited with the Korea Biotechnology Research Institute Gene Bank on June 24, 2002 and received accession number KCTC 10292BP.

The recombinant luminescent / fluorescent bacterium Escherichia coli RFM443 / pDK1, pACRG43 (EBDUAL22) [KCTC10292BP] was found to increase the fluorescence intensity when detecting a genetically damaging material, and increased the luminescence intensity when detecting an oxidatively damaging material.

Therefore, the recombinant strain according to the present invention can predict the possibility of oxidative and genetic damage of various chemicals and can sensitively detect the toxicity and harmfulness of water samples.

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 recA promoter is described in recA :: luxCDABE [Vollmer, A.C., Belkins, S., Smulski, D.R., Van Dyk, T.K., LaRosa, R.A., Appl. Environ. Microbiol. 61 (11), 4124-4127] was amplified using a PCR method (94 ℃ 1 minute, 56 ℃ 1 minute, 72 ℃ 1 minute, after 34 cycles 72 ℃ 10 minutes). The following primers were used for amplification up to -184 and +33 portions of the recA promoter.

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

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

This PCR product was treated with BamHI and KpnI restriction enzymes and recombined with the plasmid vector pDM1, which was also treated with the same enzyme, to make pDMR1. pDM1 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 combined by treatment with EcoRI and HindIII restriction enzymes, respectively. The prepared pDMR1 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. The recA :: GFPuv4 region in the pRG7 plasmid was amplified by PCR (1 min at 94 ° C, 1 min at 56 ° C, 1 min at 72 ° C, 72 min at 34 ° C after 34 cycles). Primers for PCR are as follows.

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

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

This PCR product was put into TA cloning plasmid pGEM-T Easy [Promega] to prepare pRGTA plasmid. pRTGA was treated with EcoRI restriction enzyme to obtain a recA :: GFPuv4 moiety, which was then combined with pACYC184 [New England Biolabs] which was also treated with EcoRI restriction enzyme to finally produce pACRG43 [FIG. 1].

On the other hand, pDK1 production process is as follows.

katG :: luxCDABE [Belkin, S., Smulski, D.R., Vollmer, A.C., Van Dyk, T.K., LaRossa, R.A. Appl Environ Microbiol. 62: 2252-2256], and the katG promoter was amplified using PCR (94 ° C 1 minute, 57 ° C 1 minute, 72 ° C 1 minute, 34 cycles 72 ° C 10 minutes) using the following primers.

5 'Primer: 5'- GTCCGGATCCGGACATAATCAAAAAAGC-3' (SEQ ID NO: 4)

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

The PCR product was treated with BamHI and KpnI restriction enzyme and recombined with pDEW201, which was also treated with the same restriction enzyme, to prepare pDK1 [FIG. 2].

The plasmids pDK1 and pACRG43 were confirmed to be the desired plasmid using restriction enzymes [FIG. 3]. Line 1 was treated with EcoRI for pRG7 and showed a bp band of 4000 bp. Line 2 was treated with EcoRI for pRGTA, and the following 1000 bp recA :: GFPuv4 PCR product was inserted into the pGEM-T Easy vector. Line 3 was ecoRI-treated pACRG43, confirming that recA :: GFPuv4 with EcoRI sequences at both ends was correctly inserted into pACYC184. Line 4 was treated with EcoRI simultaneously with pACRG43 and pDK1. Line 5 was treated with EcoRI for pDK1, confirming that about 10000 bp of single stranded DNA appeared.

Example 2 Preparation of EBDUAL22 Strains

The plasmids pDK1 and pACRG43 prepared in Example 1 were transformed into E. coli RFM443. The pDK1 plasmid has an ampicillin resistance gene and the pACRG43 plasmid has a tetracycline resistance gene. First, pDK1 was introduced into Escherichia coli RFM443, and colonies containing pDK1 were selected from LB medium containing ampicillin, and pACRG43 plasmid was introduced into the strain, and both pDK1 and pACRG43 plasmids were contained in LB medium containing ampicillin and tetracycline. Colonies were screened.

The luminescence / fluorescence expression of selected colonies was confirmed using a substance that induces gene damage and a substance that induces oxidative damage. Selected colonies were shaken at 37 ° C. using a 15 ml tube containing 5 ml LB medium containing 50 mg / ml ampicillin and 10 mg / ml tetracycline. The 100 μl cultured here was shaken at the same temperature in a 250 mL flask containing 100 mL of LB medium at the same concentration. At 600 nm, OD 0.08 was inoculated with hydrogen peroxide or MNNG (Methyl-N-nitro-N-nitrosoguanidine), which is a cellular oxidative damage agent. Luminescence intensity after inoculation was determined using a TD20e luminometer (Turner, USA) in culture inoculated with oxidative damage. 100 μl is taken every 30 minutes from a sample inoculated with chemicals and shaken in a mini tube, and a mini tube is put into a Turner TD20e luminometer to check luminescence intensity. Fluorescence intensity was confirmed using FL600 fluorimeter (Bio-Tek, USA) in the culture medium inoculated with MNNG. 100 μl is taken every 2 hours and transferred to a FL600 sample dish to measure the fluorescence intensity. Cell OD was measured using a UV spectrophotometer (Lambda 12, Perkin-Elmer, USA).

The transformed recombinant luminescent / fluorescent bacterium Escherichia coli RFM443 / pACRG43, pDK1 (EBDUAL22) was deposited with the Korea Biotechnology Research Institute Gene Bank on June 24, 2002 and received accession number KCTC 10292BP.

Example 3: Confirmation of Gene Damage and Oxidative Damage Detection of Transformed Strains

EBDUAL22 strains were cultured in 100 ml LB medium containing 50 mg / ml and 10 mg / ml of ampicillin and tetracycline, respectively, to an absorbance of 0.08 (600 nm), followed by 0.003% hydrogen peroxide or MNNG (Methyl-N-nitro-N). -nitrosoguanidine) was inoculated with 0.4 ppm of luminescence and fluorescence intensity for 24 hours. In the case of hydrogen peroxide inoculation, cell growth was slightly delayed, but the luminescence value increased significantly after inoculation, and the fluorescence intensity did not increase significantly. This means that hydrogen peroxide induces oxidative damage in the cell and hence katG expression.

In the case of MNNG inoculation, it was confirmed that the increase in fluorescence intensity was apparent after 7 hours compared to the control group. However, when the cell growth state was late stagnant, the value was not different from that of the control group [FIG. 4]. This means that MNNG induces genetic damage in cells and thus induces recA expression.

As described above, the recombinant strain according to the present invention serves as a detector that can provide a criterion for simultaneously analyzing the toxicity of various chemicals and water samples and classifying the hazards into gene damage and oxidative damage. It can be useful in the field of toxicity assessment of chemicals and water ecosystems.

Claims (4)

Recombinant plasmid pACRG43 produced by fusion of recA, an expression promoter of RecA protein formed by gene damage of a cell, and GFPuv4, a fluorescent protein gene. Recombinant plasmid pDK1 produced by fusion of katG, an expression promoter of KatG protein formed by oxidative damage of cells, with lux CDABE, a luminescent gene. E. coli RFM443 / pACRG43, pDK1 (EBDUAL22) [KCTC10292BP] transformed by introducing pACRG43 of claim 1 and pDK1 of claim 2 into E. coli RFM443. E. coli RFM443 / pACRG43, pDK1 (EBDUAL22) [KCTC10292BP] for simultaneous detection of genetic and oxidative damage agents.