KR100461757B1 - The recombinant bioluminescent bacteria for detection of chemicals' toxicity and harmfulness - Google Patents
The recombinant bioluminescent bacteria for detection of chemicals' toxicity and harmfulness Download PDFInfo
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- 231100000419 toxicity Toxicity 0.000 title claims abstract description 21
- 230000001988 toxicity Effects 0.000 title claims abstract description 21
- 239000000126 substance Substances 0.000 title claims abstract description 19
- 241000894006 Bacteria Species 0.000 title claims abstract description 8
- 238000001514 detection method Methods 0.000 title description 5
- 241000588724 Escherichia coli Species 0.000 claims abstract description 30
- 239000013612 plasmid Substances 0.000 claims abstract description 13
- 238000004020 luminiscence type Methods 0.000 claims abstract description 11
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 9
- 241001515965 unidentified phage Species 0.000 claims abstract description 6
- 238000000034 method Methods 0.000 claims description 3
- 238000003776 cleavage reaction Methods 0.000 claims 1
- 230000007017 scission Effects 0.000 claims 1
- 238000011156 evaluation Methods 0.000 abstract description 3
- 239000003440 toxic substance Substances 0.000 description 9
- 231100000614 poison Toxicity 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 6
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 6
- 238000005415 bioluminescence Methods 0.000 description 6
- 230000029918 bioluminescence Effects 0.000 description 6
- 229960000723 ampicillin Drugs 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 229960004857 mitomycin Drugs 0.000 description 3
- XMTQQYYKAHVGBJ-UHFFFAOYSA-N 3-(3,4-DICHLOROPHENYL)-1,1-DIMETHYLUREA Chemical compound CN(C)C(=O)NC1=CC=C(Cl)C(Cl)=C1 XMTQQYYKAHVGBJ-UHFFFAOYSA-N 0.000 description 2
- 108010054576 Deoxyribonuclease EcoRI Proteins 0.000 description 2
- 241000701959 Escherichia virus Lambda Species 0.000 description 2
- 241000607598 Vibrio Species 0.000 description 2
- 231100000045 chemical toxicity Toxicity 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 241000607568 Photobacterium Species 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
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- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229910001410 inorganic ion Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 231100000683 possible toxicity Toxicity 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 230000008791 toxic response Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
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- B01J23/16—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00 of arsenic, antimony, bismuth, vanadium, niobium, tantalum, polonium, chromium, molybdenum, tungsten, manganese, technetium or rhenium
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Abstract
본 발명은 화학물질의 독성과 그 유해성을 탐지할 수 있는 재조합 발광 박테리아에 관한 것으로서, 더욱 상세하게는 PR(박테리오파지 프로모터) 프로모터 유전자를 함유하는 재조합 플라스미드 pPRLUX를 제작하고, 이 플라스미드를 대장균에 도입하여 항시 발광성을 갖도록 형질전환시켜 화학물질의 독성 정도를 민감하게 평가할 수 있도록 한 재조합 박테리아 대장균 DH5@pPRLUX[KCTC 10240BP]에 관한 것이다.The present invention relates to a recombinant luminescent bacterium capable of detecting the toxicity and hazards of chemicals, and more particularly, to construct a recombinant plasmid pPRLUX containing a P R (bacteriophage promoter) promoter gene and introducing the plasmid into E. coli. The present invention relates to a recombinant bacterium Escherichia coli DH5 @ pPRLUX [KCTC 10240BP], which is transformed to have luminescence at all times to allow a sensitive evaluation of the toxicity of chemicals.
Description
본 발명은 화학물질의 독성과 그 유해성을 탐지할 수 있는 재조합 발광 박테리아에 관한 것으로서, 더욱 상세하게는 PR(박테리오파지 프로모터) 프로모터 유전자를 함유하는 재조합 플라스미드 pPRLUX를 제작하고, 이 플라스미드를 대장균에 도입하여 항시 발광성을 갖도록 형질전환시켜 화학물질의 독성 정도를 민감하게 평가할 수 있도록 한 재조합 박테리아 대장균 DH5@pPRLUX[KCTC 10240BP]에 관한 것이다.The present invention relates to a recombinant luminescent bacterium capable of detecting the toxicity and hazards of chemicals, and more particularly, to construct a recombinant plasmid pPRLUX containing a P R (bacteriophage promoter) promoter gene and introducing the plasmid into E. coli. The present invention relates to a recombinant bacterium Escherichia coli DH5 @ pPRLUX [KCTC 10240BP], which is transformed to have luminescence at all times to allow a sensitive evaluation of the toxicity of chemicals.
기존의 유해성 평가를 위한 발광성 박테리아는 비브리오 피쉐리(Vibrio fisheri)혹은 포토박테리움 포스포리움(Photobacterium phosporium)등이 주로 이용되어 왔다. 현재까지 이들 균주를 이용한 상용화된 독성 탐지방법에는 MICROTOX[Jennings et al., Water Res 2001 Oct; 35(14):3448-56]가 있다.Existing luminescent bacteria for hazard assessment have been mainly used such as Vibrio fisheri or Photobacterium phosporium . To date, commercialized toxicity detection methods using these strains include MICROTOX [Jennings et al., Water Res 2001 Oct; 35 (14): 3448-56.
상기 방법은 자연적인 상태에서 일정한 빛이 유지되며 독성물질과 접촉 시 대사 저해나 세포의 사멸에 의한 빛 감소를 통하여 독성을 평가하였다. 이들 균주는 해저 미생물이므로 독성 평가 시 샘플 내에 존재하는 염분 농도를 상황에 따라 조절해 주어야 하며, 염분 농도에 따라 반응 가능한 독성 물질의 농도 범위가크게 차이가 나 독성 물질 탐지가 어려웠다. 또한, 배양 조건을 위해 여러 가지 무기이온 등이 첨가되어야하며 배양된 조건에 따라서 독성 반응이 달라지는 문제점이 있다.The method maintains a constant light in a natural state and evaluated the toxicity through light reduction by metabolism inhibition or cell death upon contact with the toxic substance. Since these strains are subsea microorganisms, the salt concentration present in the sample should be adjusted according to the situation when toxicity evaluation was performed, and it was difficult to detect the toxic substances because the concentration ranges of the toxic substances that could be reacted were greatly different. In addition, various inorganic ions should be added for the culture conditions, and there is a problem in that the toxic response varies depending on the culture conditions.
이에, 본 발명자들은 상기와 같은 점을 감안하여 발광이 항시 나타나는 재조합 균주를 위해 배양 조건이 쉬운 대장균을 이용하였으며, 넓은 범위에서의 화학물질 독성을 탐지할 수 있는 탐지체로서 cI 유전자의 억제 작용이 없는 박테리오파지의 PR프로모터를 lux 오페론(operon)과 결합하여 생물학적 발광이 항시 나타나는 재조합 발광 박테리아 대장균 DH5@pPRLUX[KCTC 10240BP]을 개발함으로써 본 발명을 완성하게 되었다.In view of the above, the present inventors used E. coli, which is easy to culture conditions, for recombinant strains that exhibit luminescence at all times. As a detector capable of detecting chemical toxicity in a wide range, the inhibitory effect of the cI gene is The present invention was completed by combining the free bacteriophage P R promoter with lux operon to develop a recombinant luminescent bacterium E. coli DH5 @ pPRLUX [KCTC 10240BP], in which biological luminescence is always present.
따라서, 본 발명은 화학물질의 독성과 그 유해성으로 인해 대장균의 발광이 감소하는 원리로 다양한 화학물질의 잠재적 독성과 그 유해성을 탐지할 수 있는 재조합 발광 박테리아 대장균 DH5@pPRLUX[KCTC 10240BP]를 제공하는데 그 목적이 있다.Accordingly, the present invention provides a recombinant luminescent bacterium E. coli DH5 @ pPRLUX [KCTC 10240BP] capable of detecting the potential toxicity and the harmfulness of various chemicals on the principle that the luminescence of E. coli is reduced due to the toxicity and harmfulness of chemicals. The purpose is.
도 1은 재조합 벡터 pPRLUX의 유전자 지도와 및 PCR에 의한 삽입한 프로모터를 0.8% 아가로스 젤 전기 영동으로 확인한 사진(Lane 1: PCR product, Lane 2 : 100 bp DNA marker)을 나타낸 것이다.Figure 1 shows a genetic map of the recombinant vector pPRLUX and a picture (Lane 1: PCR product, Lane 2: 100 bp DNA marker) confirmed by 0.8% agarose gel electrophoresis of the inserted promoter by PCR.
도 2는 본 발명에 따른 재조합 발광박테리아 대장균 DH5@pPRLUX[KCTC 10240BP]과 대조군 대장균 DH5@pUCD615(Wild type)와의 생물학적 발광량을 비교한 그래프이다.Figure 2 is a graph comparing the amount of bioluminescence between recombinant E. coli DH5 @ pPRLUX [KCTC 10240BP] and the control E. coli DH5 @ pUCD615 (Wild type) according to the present invention.
도 3은 재조합 발광 박테리아 대장균 DH5@pPRLUX에 마이토마이신 C를 농도 별로 주입 시 생물학적 발광량을 나타낸 그래프이다.3 is a graph showing the amount of bioluminescence upon injection of mitomycin C by concentration into recombinant luminescent bacteria E. coli DH5 @ pPRLUX.
도 4는 재조합 발광 박테리아 대장균 DH5@pPRLUX에 페놀을 농도별로 주입 시 생물학적 발광량을 나타낸 그래프이다.4 is a graph showing the amount of bioluminescence when phenol is injected into the recombinant luminescent bacterium E. coli DH5 @ pPRLUX by concentration.
도 5는 재조합 발광 박테리아 대장균 DH5@pPRLUX에 에탄올을 농도별로 주입 시 생물학적 발광량을 나타낸 그래프이다.5 is a graph showing the amount of bioluminescence when ethanol is injected by concentration into recombinant luminescent bacterium E. coli DH5 @ pPRLUX.
도 6은 재조합 발광 박테리아 대장균 DH5@pPRLUX에 과산화수소를 농도별로주입 시 생물학적 발광량을 나타낸 그래프이다.6 is a graph showing the amount of bioluminescence when injecting hydrogen peroxide into recombinant luminescent bacteria E. coli DH5 @ pPRLUX by concentration.
본 발명은 박테리오파지의 PR프로모터 유전자를 함유하는 재조합 플라스미드 pPRLUX를 대장균에 도입하여 형질전환된 재조합 발광 박테리아 대장균DH5@pPRLUX[KCTC 10240BP] 및 이를 이용한 화학물질의 독성 및 유해성 탐지 방법을 그 특징으로 한다.The present invention is characterized by introducing the recombinant plasmid pPRLUX containing the P R promoter gene of bacteriophage into Escherichia coli, and transforming the recombinant luminescent bacterium Escherichia coli DH5 @ pPRLUX [KCTC 10240BP] and chemicals using the same. .
이와 같은 본 발명을 상세히 설명하면 다음과 같다.The present invention will be described in detail as follows.
본 발명은 다양한 화학물질의 독성을 평가할 수 있는 유전공학적으로 변형된 재조합 발광성 박테리아 대장균 DH5@pPRLUX[KCTC 10240BP]에 관한 것으로, 이 재조합 박테리아를 이용하여 지구상에 존재하는 다양한 화학물질의 독성과 그 유해성을 평가할 수 있다.The present invention relates to a genetically engineered recombinant luminescent bacterium E. coli DH5 @ pPRLUX [KCTC 10240BP] capable of evaluating the toxicity of various chemicals, and using the recombinant bacteria, the toxicity and hazards of various chemicals present on the earth. Can be evaluated.
화학물질의 독성을 탐지할 수 있는 본 발명에 따른 신균주는 발광의 항시 유도를 위해 박테리오파지의 항시 발현 유도 프로모터인 PR프로모터 부분을 생물학적 빛을 발할 수 있는 lux 오페론이 들어있는 재조합 플라스미드와 융합하고 이를 대장균에 도입하여 형질전환시켜 제작한 것이다.The new strain according to the present invention, which can detect the toxicity of chemicals, is fused with a recombinant plasmid containing lux operon capable of emitting biological light to the P R promoter portion, which is a constant expression-inducing promoter of bacteriophage, for the continuous induction of luminescence. This was produced by introducing into E. coli and transformed.
이 재조합 플라스미드를 소유한 형질 전환체로부터 발현된 콜로니 중 성장에 따라 항시 발광을 나타내는 콜로니를 최종적으로 선별하였다. 상기 형질전환된 재조합 발광 박테리아 대장균 DH5@pPRLUX를 한국생명공학연구원 유전자 은행에 2002년 5월 3일자로 기탁하여 수탁번호 KCTC 10240BP를 부여받았다.Colonies showing luminescence at all times were finally selected from the colonies expressed from the transformants carrying the recombinant plasmid. The transformed recombinant luminescent bacterium Escherichia coli DH5 @ pPRLUX was deposited with the Korea Biotechnology Research Institute Gene Bank on May 3, 2002 and received accession number KCTC 10240BP.
이러한 재조합 발광 박테리아 대장균 DH5@pPRLUX[KCTC 10240BP]는 PR프로모터의 작동에 의해 항시 발광하는 성질을 갖게되며 다양한 화학물질과 수질 샘플의 독성과 유해정도를 민감하게 탐지할 수 있다.The recombinant luminescent bacterium E. coli DH5 @ pPRLUX [KCTC 10240BP] has a property to emit light at all times by the operation of the P R promoter and can sensitively detect the toxicity and harmfulness of various chemicals and water samples.
이하, 본 발명은 다음 실시예에 의거하여 더욱 상세히 설명하겠는바, 본 발명이 이에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.
실시예 1: 화학물질 독성과 유해성 탐지를 위한 재조합 플라스미드의 제작Example 1 Construction of Recombinant Plasmids for Chemical Toxicity and Hazard Detection
대장균으로부터 분리된 박테리오파지(Bacteriophage Lambda)의 PR프로모터를 갖고 있는 pPL450[Love C.A. et al. Gene 1996 176:19-53] 플라스미드를 주형으로 하여 PR프로모터 부분에 대해서BamHI과EcoRI 제한 효소 부위를 포함하는 5'-프라이머(서열번호 1: 5'-GCCGGGATCCTTAAATCTATCACCGCAAGG)와 3'-프라이머(서열번호 2: 5'-GCCGGAATTCGATCTTTAGCTGTCTTGGTTT)를 사용하여 PCR 162 bp(서열번호 3)의 산물을 얻었다. PCR 산물은 PR프로모터의 영역을 완전히 포함하였다. 또한, PCR 반응 시 94 ℃ 1분, 58 ℃ 1분, 72 ℃ 1분의 조건으로 34번 반복 수행하였다. 상기 PCR 산물은 발광성을 나타내는 비브리오 피쉐리(Vibrio fisheri)의 lux CDABE 오페론 유전자 앞에 프로모터를 포함하지 않고 있으며, 카나마이신(Kanamycin)과 암피실린(Ampicillin) 저항성 유전자를 포함하는 벡터 pUCD615[Rogowsky et al. (J. Bacterial, 169 (11) pp 5101-5112, (1987)]와BamHI과EcoRI으로 처리한 후 연결하였다. 이 결과물을 pPRLUX라 명명하였다.PPL450 with P R promoter of Bacteriophage Lambda isolated from Escherichia coli [Love CA et al. Gene 1996 176: 19-53] 5'-primers (SEQ ID NO: 1: 5'-GCCGGGATCCTTAAATCTATCACCGCAAGG) and 3'-primers containing Bam HI and Eco RI restriction enzyme sites for the P R promoter moiety using the plasmid as a template. The product of PCR 162 bp (SEQ ID NO: 3) was obtained using SEQ ID NO: 5'-GCCGGAATTCGATCTTTAGCTGTCTTGGTTT. PCR products completely covered the region of the P R promoter. In addition, the PCR reaction was repeated 34 times under the conditions of 94 ° C 1 minute, 58 ° C 1 minute, 72 ° C 1 minute. The PCR product does not include a promoter in front of the lux CDABE operon gene of Vibrio fisheri , which exhibits luminescence, and includes a vector pUCD615 [Rogowsky et al. (J. Bacterial, 169 (11) pp 5101-5112, (1987)), followed by treatment with Bam HI and Eco RI. The resulting product was named pPRLUX.
상기 플라스미드 pPRLUX를 주형으로 위에서 사용된 프라이머 세트를 이용하여 동일 조건의 PCR[도 1]과 시퀀싱을 통해 원하는 크기의 산물이 들어갔음을 확인하였다.The plasmid pPRLUX as a template was confirmed that the product of the desired size was entered through PCR and sequencing under the same conditions using the primer set used above.
실시예 2 : 재조합 발광 박테리아 생산Example 2: Recombinant Luminescent Bacteria Production
상기 실시예 1에서 만들어진 플라스미드 pPRLUX를 대장균(MAX Efficiency DH5@ Competent cells, GIBCOBRL)에 도입하여 형질전환시켰으며, 이 pPRLUX를 소유한 세포는 그 내부의 항 암피실린 유전자의 활동으로 암피실린 선택 배지에서 생존 가능하다. 살아남은 세포들 중에서 세포 성장에 따른 생물학적 빛을 발광하는 콜로니를 선별하고, 독성 물질의 스트레스에 따라 빛의 감소가 효과적으로 감소하는 콜로니를 최종 선별하였다.The plasmid pPRLUX prepared in Example 1 was transformed by introduction into E. coli (MAX Efficiency DH5 @ Competent cells, GIBCOBRL), and cells possessing this pPRLUX were able to survive in ampicillin selection medium due to the activity of an anti-ampicillin gene therein. Do. Among the surviving cells, colonies that emit biological light according to cell growth were selected, and colonies that effectively reduced light reduction according to stress of toxic substances were finally selected.
형질전환된 재조합 발광 박테리아 대장균 DH5@pPRLUX를 한국생명공학연구원 유전자은행에 2002년 5월 3일자로 기탁하여 수탁번호 KCTC 10240BP를 부여받았다.The transformed recombinant luminescent bacterium Escherichia coli DH5 @ pPRLUX was deposited with the Korea Biotechnology Research Institute Gene Bank on May 3, 2002 and received accession number KCTC 10240BP.
실시예 3: 형질전환된 재조합 발광 박테리아 대장균 DH5@pPRLUX의 발광량 확인Example 3: Confirmation of luminescence amount of transformed recombinant luminescent bacterium E. coli DH5 @ pPRLUX
생물학적 발광량 측정은 먼저 선택된 콜로니를 30 ℃ , 250 rpm으로 쉐이킹 인큐베이터에서 암피실린을 포함하는 LB 배지 100 ㎖[DIFCO, USA]에서 600 nm에서 0.8이 될 때까지 배양한 후, 이중 2 ㎖을 LB 배지 100 ㎖ 넣어 위와 동일한 조건으로 진탕 배양하면서 발광량을 DH5@pUCD615(대조군)와 비교하였다. 비교 결과 배양 과정 중 pPRLUX가 도입된 DH5@pPRLUX[KCTC 10240BP]의 발광량이 대조군에 비해 16000배(도 2의 y축 값은 순수 발광 값) 까지 증가하는 것을 확인하였다[도 2].The bioluminescence measurement was carried out by first incubating the selected colonies in a shaking incubator at 30 ° C. and 250 rpm until 100 0.8 at 600 nm in 100 ml of LB medium containing Ampicillin [DIFCO, USA], followed by 2 ml of LB medium 100 The amount of luminescence was compared with DH5 @ pUCD615 (control) while shaking and cultured under the same conditions as the mL. As a result, it was confirmed that the luminous amount of DH5 @ pPRLUX [KCTC 10240BP] into which pPRLUX was introduced was increased by 16000 times (y-axis value of FIG. 2 was pure emission value) compared to the control group [FIG. 2].
실시예 4: 재조합 발광 박테리아 대장균 DH5@pPRLUX[KCTC 10240BP]의 독성 물질에 대한 탐지능 확인Example 4 Determination of the Detection of Recombinant Luminescent Bacteria Escherichia Coli DH5 @ pPRLUX [KCTC 10240BP] for Toxic Substances
선택된 콜로니를 30 ℃, 250 rpm으로 쉐이킹 인큐베이터에서 암피실린( ampicillin)을 200 ㎍/㎖ 포함하는 LB 배지 100 ㎖[DIFCO, USA]에서 600 nm에서 O.D. 0.8이 될 때까지 배양한 후, 이중 2 ㎖을 LB 배지 100 ㎖ 넣어 배양하고 이를 다시 O.D. 1.0 까지 배양하였다. 그런 다음 재조합 발광 박테리아 대장균 DH5@pPRLUX(EBBC1) 150 ㎕을 96 웰 플레이트[Microtiter TM, DYNEX technologies, USA] 에 넣고 다양한 농도의 독성 물질을 150 ㎕ 주입한 후 30 ℃ 에서 96 웰 루미노미터[DYNEX technologies, USA]를 이용하여 4시간 동안 10분 간격으로 측정하였다.Selected colonies were 30 ° C. at 250 rpm in a shaking incubator with 200 μg / ml of ampicillin in 100 ml of LB medium [DIFCO, USA] at 600 nm at 600 nm. After incubation until 0.8, 2 ml of this was added to 100 ml of LB medium and cultured again. Incubated to 1.0. Then, 150 µl of recombinant luminescent bacterium Escherichia coli DH5 @ pPRLUX (EBBC1) was put into a 96 well plate [MicrotiterTM, DYNEX technologies, USA], and 150 µl of various concentrations of toxic substances were injected, followed by a 96-well luminometer [DYNEX] at 30 ° C. technologies, USA] were measured at 10 minute intervals for 4 hours.
측정에 사용된 독성 물질로는 마이토마이신 C, 페놀, 에탄올, 과산화수소수로 이들의 농도는 다음과 같다.Toxic substances used in the measurement are mitomycin C, phenol, ethanol and hydrogen peroxide. Their concentrations are as follows.
마이토마이신 C : 10 ppm, 5 ppm, 2.5 ppm, 1.25 ppm, 0.625 ppm, 0.3125 ppm, 0.15625 ppm, 0.078125 ppm, 0.0390625 ppmMitomycin C: 10 ppm, 5 ppm, 2.5 ppm, 1.25 ppm, 0.625 ppm, 0.3125 ppm, 0.15625 ppm, 0.078125 ppm, 0.0390625 ppm
페놀 : 1000 ppm, 500 ppm, 250 ppm, 125 ppm, 62.5 ppm, 31.25 ppm, 15.625 ppm, 7.8125 ppm, 3.90625 ppmPhenol: 1000 ppm, 500 ppm, 250 ppm, 125 ppm, 62.5 ppm, 31.25 ppm, 15.625 ppm, 7.8125 ppm, 3.90625 ppm
에탄올 : 5%, 2.5%, 1.25%, 0.625%, 0.3125%, 0.15625%, 0.078125%, 0.0390625%, 0.019053125 %Ethanol: 5%, 2.5%, 1.25%, 0.625%, 0.3125%, 0.15625%, 0.078125%, 0.0390625%, 0.019053125%
과산화수소 : 1%, 0.5%, 0.25%, 0.125%, 0.0625%, 0.03125%, 0.015625%, 0.0078125%, 0.00390625%, 0.0019053125%Hydrogen peroxide: 1%, 0.5%, 0.25%, 0.125%, 0.0625%, 0.03125%, 0.015625%, 0.0078125%, 0.00390625%, 0.0019053125%
생물학적 빛의 단위는 BL(Bioluminescnent level)을 사용하며 발광량의 감소정도는 독성 물질을 주입한 경우의 발광량(sample bioluminescent level)과 독성물질을 주입하지 않은 경우의 발광량(control bioluminescent level)을 비교하여 다음 수학식 1을 이용하여 상대적 발광량 값(Relative bioluminescent level, RBL)을 구하여 화학물질의 독성 정도를 평가할 수 있다. 독성이 강할수록 상대적 발광량 값이 작아진다. 상대 발광량 값은 측정 후 2시간의 값을 사용하였다.The unit of biological light uses BL (Bioluminescnent level), and the decrease in the amount of light emission is compared with the sample bioluminescent level when toxic substances are injected and the control bioluminescent level when no toxic substances are injected. Equation 1 can be used to evaluate the degree of toxicity of a chemical by obtaining a relative bioluminescent level (RBL). The stronger the toxicity, the smaller the value of relative light emission. The relative light emission value used was a value of 2 hours after the measurement.
도 3 내지 도 6에 나타낸 바와 같이, 화학물질의 경우 특정 농도 이상에서부터 빛 감소 현상이 나타나며 농도가 증가할수록 빛의 감소가 증가하는 경향이 두드러지게 나타났다.As shown in FIGS. 3 to 6, in the case of chemicals, light decrease occurs from a certain concentration or more, and the decrease in light increases as the concentration increases.
이상에서 설명한 바와 같이, 본 발명에 따른 재조합 발광 박테리아 대장균 DH5@pPRLUX[KCTC10240BP]은 다양한 화학물질과 수질 샘플의 독성 분석과 그 유해성을 탐지하고 평가할 수 있는 기준을 제시할 수 있는 탐지체 역할을 하며, 화학물질 및 수질 생태계의 독성 평가 분야에서 유용하게 사용될 수 있다.As described above, the recombinant luminescent bacterium Escherichia coli DH5 @ pPRLUX [KCTC10240BP] according to the present invention serves as a detector capable of providing a standard for detecting and evaluating toxicity and analyzing the toxicity of various chemicals and water samples. This can be useful in the field of toxicity assessment of chemicals and water ecosystems.
<110> Kwang Ju Institute of Science and Technology <120> The recombinant bioluminescent bacteria for detection of chemicals' toxicity and harmfulness <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> 5'-PRIMER <400> 1 gccgggatcc ttaaatctat caccgcaagg 30 <210> 2 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> 3'-PRIMER <400> 2 gccggaattc gatctttagc tgtcttggtt t 31 <210> 3 <211> 162 <212> DNA <213> Bacteriophage lambda <400> 3 ggatccttaa atctatcacc gcaaggcacc gtgcgtgttg actttttacc tctggcggtg 60 ataatggttg catgtactaa ggaggttgta tggaacaacg cataaccctg aaagattatg 120 caatgcgctt tgggcaaacc aagacagcta aagatcgaat tc 162<110> Kwang Ju Institute of Science and Technology <120> The recombinant bioluminescent bacteria for detection of chemicals' toxicity and harmfulness <160> 3 <170> Kopatent In 1.71 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> 5'-PRIMER <400> 1 gccgggatcc ttaaatctat caccgcaagg 30 <210> 2 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> 3'-PRIMER <400> 2 gccggaattc gatctttagc tgtcttggtt t 31 <210> 3 <211> 162 <212> DNA <213> Bacteriophage lambda <400> 3 ggatccttaa atctatcacc gcaaggcacc gtgcgtgttg actttttacc tctggcgggg aatcatc
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| US5589337A (en) * | 1992-07-06 | 1996-12-31 | The President And Fellows Of Harvard College | Methods and diagnostic kits for determining toxicity utilizing bacterial stress promoters fused to reporter genes |
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| KR20000029543A (en) * | 1996-07-29 | 2000-05-25 | 릿지 리 | Biosensors |
| KR20020041603A (en) * | 2000-11-28 | 2002-06-03 | 박호군 | Use of plasmid containing luxCDABE and recombinant E. coli SB01 which was transformed by this plasmid |
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| US5589337A (en) * | 1992-07-06 | 1996-12-31 | The President And Fellows Of Harvard College | Methods and diagnostic kits for determining toxicity utilizing bacterial stress promoters fused to reporter genes |
| US5731163A (en) * | 1994-11-23 | 1998-03-24 | E. I. Du Pont De Nemours And Company | Lyophilized bioluminescent bacterial reagent for the detection of toxicants |
| KR20000029543A (en) * | 1996-07-29 | 2000-05-25 | 릿지 리 | Biosensors |
| KR20020041603A (en) * | 2000-11-28 | 2002-06-03 | 박호군 | Use of plasmid containing luxCDABE and recombinant E. coli SB01 which was transformed by this plasmid |
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