KR100473350B1 - Recombinant bioluminescent bacteria for the detection oxidative damage - Google Patents
Recombinant bioluminescent bacteria for the detection oxidative damage Download PDFInfo
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Abstract
본 발명은 산화적 손상을 탐지하는 재조합 발광박테리아에 관한 것으로서, 더욱 상세하게는 과산화수소는 물론 파라코트(Paraquat) 등의 산화적 손상을 주는 물질에 민감하게 반응하는 Pqi-5 단백질의 pqi-5 프로모터와 발광유전자를 결합하여 재조합 플라스미드 pPQI5를 제작하고, 이 플라스미드를 대장균에 도입하여 세포의 산화적 손상을 유도하는 물질을 탐지하게 될 경우 발광성이 증가하도록 형질 전환하여, 리포터 유전자의 종류에 따라 화학물질의 독성 정도를 산화적 손상으로 구분하여 민감하게 평가할 수 있도록 한 재조합 박테리아에 관한 것이다.The present invention relates to a recombinant luminescent bacterium that detects oxidative damage. More specifically, the pqi-5 promoter of Pqi-5 protein that reacts sensitively to oxidatively damaging substances such as hydrogen peroxide as well as paracoats. And recombinant plasmid pPQI5 was prepared by combining with the luminescent gene, and when the plasmid was introduced into Escherichia coli to detect a substance that induces oxidative damage to cells, the luminescence was transformed to increase luminescence. It relates to a recombinant bacterium that can be sensitively evaluated by classifying the toxicity level of the oxidative damage.
Description
본 발명은 산화적 손상을 탐지하는 재조합 발광박테리아에 관한 것으로서, 더욱 상세하게는 과산화수소는 물론 파라코트(Paraquat) 등의 산화적 손상을 주는 물질에 민감하게 반응하는 Pqi-5 단백질의 pqi-5 프로모터와 발광유전자를 결합하여 재조합 플라스미드 pPQI5를 제작하고, 이 플라스미드를 대장균에 도입하여 세포의 산화적 손상을 유도하는 물질을 탐지하게 될 경우 발광성이 증가하도록 형질 전환하여, 리포터 유전자의 종류에 따라 화학물질의 독성 정도를 산화적 손상으로 구분하여 민감하게 평가할 수 있도록 한 재조합 박테리아에 관한 것이다.The present invention relates to a recombinant luminescent bacterium that detects oxidative damage. More specifically, the pqi-5 promoter of Pqi-5 protein that reacts sensitively to oxidatively damaging substances such as hydrogen peroxide as well as paracoats. And recombinant plasmid pPQI5 was prepared by combining with the luminescent gene, and when the plasmid was introduced into Escherichia coli to detect a substance that induces oxidative damage to cells, the luminescence was transformed to increase luminescence. It relates to a recombinant bacterium that can be sensitively evaluated by classifying the toxicity level of the oxidative damage.
기존의 발광성 박테리아 대부분은 자연적인 상태에서 일정한 빛을 유지하다가 독성물질에 노출시 대사의 저해나 사멸의 정도에 의한 빛의 감소를 통하여 독성을 평가하였다[Microbics Corp., Carlsbad, Calif.].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.].
산화적 손상은 활성산소종인 자유 라디칼의 형태로 세포막, 단백질, 핵산 등의 생체 고분자를 변형시킴으로써 생태계를 이루는 미생물, 식물, 동물에 심각한 손상을 주며, 또한 인간에 있어서도 알츠하이머와 같은 퇴행성 정신질환, 당뇨병 그리고 노화와 여러 종류의 암에 이르는 치명적인 질환을 유발한다. 따라서, 이러한 산화적 손상을 야기시키는 자연적·인위적인 유해 독성물질에 대한 위해도 평가가 절실히 요구되는 실정이다. 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 risk assessment of natural and artificial harmful toxic substances causing such oxidative damage.
현재까지 발광박테리아를 이용한 가장 상용화된 독성의 탐지방법은 마이크로톡스(Microtox)[Jennings et al.,Water Res 2001 Oct;35(14):3448-56]인데, 이는 유해성물질에 노출된 미생물의 사멸정도에 의한 평가이므로 산화적 손상과 같은 특정 스트레스에 대한 반응성을 나타낼 수 없는 단점이 있다. To date, the most commonly used method of detecting toxic substances using luminescent bacteria is Microtox [Jennings et al., Water Res 2001 Oct; 35 (14): 3448-56], which kills microorganisms exposed to harmful substances. Because of the degree of evaluation, there is a disadvantage in that it cannot exhibit responsiveness to specific stresses such as oxidative damage.
한편, 본 발명자에 의해 대한민국 특허 출원된 제 2001-0071591호(2001. 11.17)에서는 산화적 손상에 대한 유해성 탐지를 위한 재조합 발광박테리아 대장균 DH5α/pSodaLux(EBHJ1)[KCTC 10098BP]를 개시한 바 있으나, sodA 프로모터를 사용하여 광범위한 부분에서의 유해성 탐지가 사실상 불가능한 문제점이 있다.On the other hand, Korean Patent Application No. 2001-0071591 (2001. 11.17) filed by the present inventors in the recombinant light-emitting bacterium Escherichia coli for the detection of harmful to oxidative damage Although DH5α / pSodaLux (EBHJ1) [KCTC 10098BP] has been disclosed, there is a problem that it is virtually impossible to detect hazards in a wide range using the sodA promoter.
또한, 대한민국 특허 출원된 출원번호 제 2002-0057633호에서는 산화적 손상 물질을 탐지할 수 있도록 산화적 손상에 의해 발현이 증가하는 KatG 단백질의 katG 프로모터를 발광 유전자인 lux 오페론과 결합하여 제작한 pDK1 플라스미드를 제시한 바 있으나, 상기 katG 프로모터는 과산화수소에만 특이적으로 반응하여 전반적인 산화적 손상을 포함할 수 없었던 단점이 있었다.In addition, Korean Patent Application No. 2002-0057633 discloses a pDK1 plasmid produced by combining a katG promoter of KatG protein, whose expression is increased by oxidative damage, with lux operon, a luminescent gene, to detect oxidative damage. Although it was suggested, the katG promoter had a disadvantage in that it could not include the overall oxidative damage in response to hydrogen peroxide specifically.
따라서, 과산화수소 뿐만 아니라 전반적인 산화적 손상 물질을 탐지할 수 있는 방법이 필요하다.Therefore, there is a need for a method capable of detecting not only hydrogen peroxide but also an entire oxidative damaging substance.
이에, 본 발명자들은 상기 문제점을 해결하기 위하여 연구한 결과, 또 다른 산화적 손상을 주는 화학물질로 알려진 파라코트(Paraquat)에 민감하게 반응할 뿐만 아니라 여러 가지 형태의 활성산소족에 민감한 반응을 하는 Pqi-5 단백질의 pqi-5 프로모터와 발광유전자를 결합하여 재조합 플라스미드 pPQI5를 제작하고, 이 플라스미드를 대장균에 도입하여 세포의 산화적 손상을 유도하는 물질을 탐지하게 될 경우 발광성이 증가하도록 형질전환하여 다양한 종류의 산화적 손상을 탐지할 수 있는 재조합 발광박테리아 대장균 DH5@/pPQI5 (EBJM1) [KCTC 10407BP]를 제작함으로써 본 발명을 완성하게 되었다.Accordingly, the present inventors have studied to solve the above problems, and as a result, Pqi reacts not only to Paraquat, which is known as another oxidatively damaging chemical, but also to various types of reactive oxygen species. The recombinant plasmid pPQI5 was prepared by combining the pqi-5 promoter of the -5 protein and the luminescent gene, and when the plasmid was introduced into E. coli to detect a substance that induces oxidative damage to cells, the plasmid was transformed to increase luminescence. The present invention was completed by constructing a recombinant luminescent bacterium E. coli DH5 @ / pPQI5 (EBJM1) [KCTC 10407BP] capable of detecting oxidative damage of a kind.
따라서, 본 발명은 화학물질 혹은 수질의 산화적 손상 물질을 탐지하고 그 독성의 유해성을 평가할 수 있는 재조합 발광 박테리아 대장균 DH5@/pPQI5 (EBJM1) [KCTC 10407BP]를 제공하는데 그 목적이 있다. Accordingly, an object of the present invention is to provide a recombinant luminescent bacterium E. coli DH5 @ / pPQI5 (EBJM1) [KCTC 10407BP] capable of detecting chemical or water oxidatively damaging substances and evaluating the harmfulness thereof.
본 발명은 pqi-5::luxCDABE 유전자를 포함하는 재조합 플라스미드 pPQI5 및 이 플라스미드를 대장균 DH5@에 도입하여 형질 전환된 재조합 발광 박테리아 대장균 DH5@/pPQI5(EBJM1)[KCTC 10407BP]을 그 특징으로 한다.The present invention is characterized by the recombinant plasmid pPQI5 comprising the pqi-5 :: luxCDABE gene and the recombinant luminescent bacterium E. coli DH5 @ / pPQI5 (EBJM1) [KCTC 10407BP] transformed by introducing the plasmid into E. coli DH5 @.
또한, 상기 재조합 균주를 이용한 산화적 손상 물질의 독성과 그 유해성 탐지 방법을 포함한다.In addition, the method includes a method for detecting toxicity and toxicity of an oxidatively damaging substance using the recombinant strain.
이와 같은 본 발명을 더욱 상세히 설명하면 다음과 같다.Referring to the present invention in more detail as follows.
본 발명은 과산화수소는 물론 파라코트(Paraquat) 등의 산화적 손상을 주는 물질에 민감하게 반응하는 Pqi-5 단백질의 pqi-5 프로모터와 발광유전자를 결합하여 재조합 플라스미드 pPQI5을 제작하고, 이 플라스미드를 대장균에 도입하여 세포의 산화적 손상을 유도하는 물질을 탐지하게 될 경우 발광성이 증가하도록 형질 전환하여, 리포터 유전자의 종류에 따라 화학물질의 독성 정도를 산화적 손상으로 구분하여 민감하게 평가할 수 있도록 한 재조합 박테리아에 관한 것이다.The present invention provides a recombinant plasmid pPQI5 by combining a pqi-5 promoter and a luminescent gene of Pqi-5 protein which are sensitive to oxidatively damaging substances such as hydrogen peroxide as well as paraquat. When it detects a substance that induces oxidative damage to cells, it is transformed to increase the luminescence, and the recombination allows sensitive evaluation by classifying the toxicity of chemicals into oxidative damage according to the type of reporter gene. It is about bacteria.
화학물질의 독성을 탐지할 수 있는 본 균주는 세포의 산화적 손상에 의해 형성되는 Pqi-5 단백질의 발현 프로모터인 pqi-5와 발광 유전자인 lux CDABE를 융합한 플라스미드인 pPQI5를 형질전환시켜 제작한 것이다.This strain, which can detect the toxicity of chemicals, was produced by transforming pPQI5, a plasmid fused with pqi-5, an expression promoter of Pqi-5 protein, formed by oxidative damage of cells, and lux CDABE, a luminescent gene. will be.
본 발명의 재조합 발광박테리아를 제작하기 위하여 기존의 재조합 발광박테리아 제작과정과는 달리 pqi-5 프로모터 부분의 사이즈를 크게 하여 pqi-5 프로모터와 발광 유전자의 결합을 용이하게 했을 뿐 아니라 pqi-5 프로모터와 발광 유전자의 결합으로 생성된 재조합 플라스미드의 성공여부를 PCR을 사용하여 확인하지 않고 단순히 제한효소로 잘라 확인할 수 있는 장점이 있다. 또한, 기존의 sod A와는 달리 유전자의 특성상 pqi-5는 산화적 손상을 발현시키는 soxRS 레귤론(regulon) 뿐만 아니라 단백질 생성에 관련된 요소를 발현시키는 rpoS 유전자 또한 pqi-5를 발현시킨다고 알려져 있어 보다 광범위한 부분에서 유해성을 탐지 가능하다.In order to manufacture the recombinant luminescent bacteria of the present invention, unlike the conventional recombinant luminescent bacteria manufacturing process, the size of the pqi-5 promoter was increased to facilitate the coupling of the pqi-5 promoter and the luminescent gene as well as the pqi-5 promoter. The success of the recombinant plasmid generated by the binding of the luminescent gene has the advantage that it can be confirmed by simply cutting with restriction enzymes without using PCR. In addition, unlike conventional sod A, pqi-5 is known to express pqi-5 as well as soxRS regulon, which expresses oxidative damage, as well as rpoS gene, which expresses factors related to protein production. In this area, hazards can be detected.
상기 재조합 플라스미드를 소유한 형질전환체로부터 발현된 콜로니를 선별하고 산화적 손상 물질을 이용하여 발광 감도의 변화를 확인하였다. 상기 형질 전환된 재조합 발광 박테리아 대장균 DH5@/pPQI5(EBJM1)를 한국 생명공학연구원 유전자 은행에 2002년 12월 27일자로 기탁하여 수탁번호 KCTC 10407BP를 부여받았다. 이러한 재조합 발광 박테리아 대장균 DH5@/pPQI5(EBJM1)[KCTC 10407BP]는 산화적 손상 물질을 감지할 경우 발광 강도가 증가한다. 따라서, 본 균주를 이용하여 다양한 화학물질의 산화적 손상 가능성을 예측할 수 있으며 수질 샘플의 독성과 그 유해정도를 민감하게 탐지할 수 있다. The colonies expressed from the transformants possessing the recombinant plasmids were selected and the change in luminescence sensitivity was confirmed using an oxidatively damaging substance. The transformed recombinant luminescent bacterium Escherichia coli DH5 @ / pPQI5 (EBJM1) was deposited with the Korea Biotechnology Research Institute Gene Bank on Dec. 27, 2002 and received accession number KCTC 10407BP. The recombinant luminescent bacterium E. coli DH5 @ / pPQI5 (EBJM1) [KCTC 10407BP] increases the luminescence intensity when it detects oxidative damage. Therefore, this strain can be used to predict the possibility of oxidative damage of various chemicals and to sensitively detect the toxicity and harmfulness of water samples.
이하, 본 발명은 다음 실시 예에 의거하여 상세히 설명하겠는 바, 본 발명이 이에 한정되는 것은 아니다. Hereinafter, the present invention will be described in detail based on the following examples, but the present invention is not limited thereto.
실시예 1: 재조합 플라스미드의 제작 Example 1 Construction of Recombinant Plasmids
pqi-5 프로모터는 대장균 DH5@의 게놈(Genomic) DNA로부터 PCR 방법을 이용하여 증폭하였다. -339 부분과 +343 부분까지 증폭을 위해 다음의 프라이머를 사용하였다. The pqi-5 promoter was amplified from the genomic DNA of Escherichia coli DH5 @ using a PCR method. The following primers were used for amplification up to -339 and +343.
5'-프라이머: 5'-TAGGGATCCTCAAACCAAG-3'(서열번호 1)5'-primer: 5'-TAGGGATCCTCAAACCAAG-3 '(SEQ ID NO: 1)
3'-프라이머: 5'-CTAGAATTCGCACATTGGTA-3'(서열번호 2)3'-primer: 5'-CTAGAATTCGCACATTGGTA-3 '(SEQ ID NO: 2)
이 PCR 산물을 BamHI 과 EcoR1 제한효소로 처리하고 플라스미드 벡터 pUCD615를 역시 같은 제한효소로 잘라내었다. 이것을 14 ℃에서 8시간 라이가아제를 이용하여 재조합 pPQI5를 제작하였다[도 1]. This PCR product was treated with BamHI and EcoR1 restriction enzymes and the plasmid vector pUCD615 was also cut with the same restriction enzyme. This was produced recombinant pPQI5 using ligase at 14 ° C. for 8 hours [FIG. 1].
이를 DH5@에 형질전환하여 DH5@/pPQI5(EBJM1)을 카나마이신이 포함된 아가 플레이트에 키워 선별된 콜로니를 카나마이신이 포함된 LB 배지에서 밤새 키운 후 pPQI5 플라스미드를 뽑아내었다. 이것을 BamH1 과 EcoR1 제한효소를 이용하여 잘라내 0.8% 아가로즈 젤에 걸어 나온 DNA의 크기로 원하는 프로모터 부위와 벡터가 들어있는 플라스미드가 제작되었음을 확인하였다[도 2]. This was transformed into DH5 @, and DH5 @ / pPQI5 (EBJM1) was grown on agar plates containing kanamycin, and selected colonies were grown overnight in LB medium containing kanamycin and pPQI5 plasmid was extracted. This was cut using BamH1 and EcoR1 restriction enzymes to confirm that the plasmid containing the desired promoter site and the vector was produced with the size of the DNA hung on the 0.8% agarose gel [FIG. 2].
실시예 2: EBJM1 균주의 제작 Example 2: Preparation of EBJM1 Strains
상기 실시예 1에서 만들어진 플라스미드 pPQI5를 대장균 DH5@에 도입하여 형질전환시켰다. 플라스미드 pPQI5는 암피실린과 카나마이신 저항성 유전자를 가지고 있다. pPQI5를 DH5@에 도입시켜 카나마이신이 들어있는 LB 배지에서 pPQI5가 들어간 콜로니를 선별하였다. 선별된 콜로니의 형광 발현을 세포의 산화적 손상을 유도하는 물질을 이용하여 확인하였다. The plasmid pPQI5 prepared in Example 1 was introduced into E. coli DH5 @ and transformed. Plasmid pPQI5 has ampicillin and kanamycin resistance genes. pPQI5 was introduced into DH5 @ to select colonies containing pPQI5 in LB medium containing kanamycin. Fluorescence expression of selected colonies was confirmed using a substance that induces oxidative damage of cells.
형질전환된 재조합 발광 박테리아 대장균 DH5@/pPQI5(EBJM1)를 한국생명공학연구원 유전자은행에 2002년 12월 27일자로 기탁하여 수탁번호 KCTC 10407BP를 부여받았다. The transformed recombinant luminescent bacterium Escherichia coli DH5 @ / pPQI5 (EBJM1) was deposited with the Korea Biotechnology Research Institute Gene Bank on Dec. 27, 2002 and was assigned accession number KCTC 10407BP.
실시예 3: 형질전환된 재조합 발광 박테리아 대장균 DH5@/pPQI5(EBJM1)[KCTC 10407BP]의 산화적 손상 물질 탐지능 확인 Example 3: Confirmation of oxidatively damaging ability of transformed recombinant luminescent bacterium Escherichia coli DH5 @ / pPQI5 (EBJM1) [KCTC 10407BP]
EBJM1 균주를 카나마이신이 포함된 100 ㎖ LB 배지에서 흡광도 0.08 (600nm) 까지 배양한 후 96 웰 루미노미터(Luminometer)에서 파라코트(Methyl viogen)를 1000 ppm부터 1 ppm까지 되도록 접종한 것과 과산화수소 0.00028125%부터 0.018%까지가 되도록 접종한 후 10시간동안 발광 강도를 측정하였다. 파라코트 접종의 경우 파라코트 접종 후 발광 값이 계속적으로 증가하는 것을 알 수 있었다. 또한, 최대 RBL(Relative Bioluminescence)값이 접종한 농도에 따라 증가하는(dose-dependant) 반응을 보이는 것을 알 수 있었다. 이는 파라코트 유도가능(inducible)한 pqi-5 프로모터가 파라코트에 의해 발현이 유도된 것을 의미한다[도 3]. 과산화수소 접종의 경우에는 파라코트와 마찬가지로 발광 값이 증가하는 것을 볼 수 있고 그에 따른 반응도 접종한 농도에 따라 증가하는(dose-dependant) 것을 알 수 있었다[도 4]. 이는 다른 세포의 산화적 반응에 발광하는 다른 대장균보다 더욱더 민감하게 반응하는 것을 알 수 있었다. EBJM1 strains were cultured in 100 ml LB medium containing kanamycin to absorbance of 0.08 (600 nm), and then inoculated from 100 ppm to 1 ppm of para-coat (Methyl viogen) in 96 well luminometer (0.00028125%). Luminous intensity was measured for 10 hours after inoculation to 0.018%. In the case of paracoat inoculation, it was found that the luminescence value increased continuously after the paracoat inoculation. In addition, it was found that the maximum RBL (Relative Bioluminescence) value showed a dose-dependant response with the inoculated concentration. This means that expression of the paracoat inducible pqi-5 promoter was induced by paracoat [FIG. 3]. In the case of hydrogen peroxide inoculation, it can be seen that the luminescence value is increased similarly to the paracoat, and the response is also increased depending on the inoculated concentration (dose-dependant) [FIG. This was found to be more sensitive to the oxidative reaction of other cells than other E. coli luminescent.
실시예 4: 종래 발광박테리아와 비교Example 4 Comparison with Conventional Luminescent Bacteria
같은 soxRS 레귤론에 의해 발현되는 sodA 유전자의 프로모터를 사용한 발광 박테리아인 대장균 DH5α/pSodaLux(EBHJ1), 본 발명의 대장균 DH5@/pPQI5(EBJM1)와 대표적인 산화적 손상 물질인 과산화수소를 사용하여 0.00028125%부터 0.018%까지 발광 강도를 비교 실험하였다.E. coli DH5α / pSodaLux (EBHJ1), a luminescent bacterium using the promoter of the sodA gene expressed by the same soxRS regulator, and E. coli DH5 @ / pPQI5 (EBJM1) of the present invention and 0.00028125% using a representative oxidative damaging substance The luminescence intensity was compared by 0.018%.
도 5에서 나타내는 것과 같이 대장균 DH5α/pSodaLux(EBHJ1)과 대장균 DH5@/pPQI5(EBJM1) 모두 과산화수소에서 접종한 농도의 정도에 따라 반응이 증가하는 것을 알 수 있었다. 그러나, 대장균 DH5α/pSodaLux(EBHJ1)의 발광 강도는 10인데 반해 대장균 DH5@/pPQI5(EBJM1)는 12로 발광 강도가 더 높고, 반응하는 과산화수소의 농도 또한 대장균 DH5α/pSodaLux(EBHJ1)는 0.009% 인데 반해 대장균 DH5@/pPQI5(EBJM1)은 0.00225%로 더 민감하게 반응하는 것을 알 수 있다.As shown in FIG. 5, both E. coli DH5α / pSodaLux (EBHJ1) and E. coli DH5 @ / pPQI5 (EBJM1) were found to increase in response to the degree of concentration inoculated in hydrogen peroxide. However, E. coli DH5α / pSodaLux (EBHJ1) has a luminescence intensity of 10, whereas E. coli DH5 @ / pPQI5 (EBJM1) has a luminescence intensity of 12, and the concentration of reactant hydrogen peroxide is also 0.009% of E. coli DH5α / pSodaLux (EBHJ1). In contrast, E. coli DH5 @ / pPQI5 (EBJM1) is more sensitive to 0.00225%.
이상에서 설명한 바와 같이, 본 발명에 의해 개발된 재조합 발광 박테리아 대장균 DH5@/pPQI5(EBJM1)[KCTC 10407BP]은 다양한 화학물질과 수질 샘플의 독성 분석과 그 유해성을 산화적 손상으로 분류할 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 E. coli DH5 @ / pPQI5 (EBJM1) [KCTC 10407BP] developed by the present invention is a criterion that can classify the toxicity of various chemicals and water samples and classify the hazards as oxidative damage. It acts as a detector to suggest and can be useful in the field of toxicity assessment of chemicals and water ecosystems.
도 1은 재조합 플라스미드 pPQI5의 제작과정을 나타낸 모식도이다. 1 is a schematic diagram showing the manufacturing process of the recombinant plasmid pPQI5.
도 2는 0.8% 아가로스 젤 전기 영동으로 제한효소로 잘라내어 재조합 플라스미드를 확인한 사진을 나타낸 것이다[Lane 1 : Lambda HindIII Marker, Lane 2 : pPQI5/EcoR1 and BamH1].Figure 2 is a 0.8% agarose gel electrophoresis was cut with a restriction enzyme confirmed the recombinant plasmid [Lane 1: Lambda HindIII Marker, Lane 2: pPQI5 / EcoR1 and BamH1].
도 3은 파라코트(Paraquat, Methyl viogen)를 1000 ppm부터 1 ppm까지 되도록 접종한 후 10시간동안 발광 강도를 측정한 것이다.Figure 3 is a paracoat (Paraquat, Methyl viogen) was inoculated to 1000 ppm to 1 ppm after measuring the luminescence intensity for 10 hours.
도 4는 과산화수소 0.00028125%부터 0.018%까지가 되도록 접종한 후 10시간동안 발광 강도를 측정한 것이다.FIG. 4 shows the luminescence intensity for 10 hours after inoculation to 0.00028125% to 0.018% hydrogen peroxide.
도 5는 대장균 DH5α/pSodaLux(EBHJ1)와 대장균 DH5@/pPQI5(EBJM1)의 과산화수소에 대한 민감도를 비교한 것이다[a: 대장균 DH5α/pSodaLux(EBHJ1), b: 대장균 DH5@/pPQI5(EBJM1)].FIG. 5 compares the sensitivity of E. coli DH5α / pSodaLux (EBHJ1) and E. coli DH5 @ / pPQI5 (EBJM1) to hydrogen peroxide [a: E. coli DH5α / pSodaLux (EBHJ1), b: E. coli DH5 @ / pPJI5 (E) .
<110> KwangJu Institute of Science and Technology <120> Recombinant bioluminescent bacteria for the detection oxidative damage <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid used as 5'-primer of PCR for amplifying pqi-5 promoter <400> 1 tagggatcct caaaccaag 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid used as 3'-primer of PCR for amplifying pqi-5 promoter <400> 2 ctagaattcg cacattggta 20<110> KwangJu Institute of Science and Technology <120> Recombinant bioluminescent bacteria for the detection oxidative damage <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid used as 5'-primer of PCR for amplifying pqi-5 promoter <400> 1 tagggatcct caaaccaag 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid used as 3'-primer of PCR for amplifying pqi-5 promoter <400> 2 ctagaattcg cacattggta 20
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