CN102371042A - Method for degrading a plurality of organic pollutants in environment with high efficiency by green fluorescent protein labeled microbes - Google Patents
Method for degrading a plurality of organic pollutants in environment with high efficiency by green fluorescent protein labeled microbes Download PDFInfo
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- CN102371042A CN102371042A CN2010102512792A CN201010251279A CN102371042A CN 102371042 A CN102371042 A CN 102371042A CN 2010102512792 A CN2010102512792 A CN 2010102512792A CN 201010251279 A CN201010251279 A CN 201010251279A CN 102371042 A CN102371042 A CN 102371042A
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Abstract
The invention relates to a method for degrading a plurality of organic pollutants in environment with high efficiency by green fluorescent protein labeled microbes, which belongs to the microbe technical field. The method comprises the following steps: a plasmid pGEM-GFP and a vector pK18 are performed double digestion and connected to obtain a vector plasmid pK-GFP-2, turned into Escherichia coli and amplified to extract plasmid, a GFP fragment is obtained by digestion, the amplified product of the recovered GFP fragment is connected with digestion pTOPO17beta-HSD-600-3 to obtain a recombined plasmid, the recombined plasmid enters in C.T competent cells through electrotransformation, and obtains a modified strain C.T17beta-GFP by screening; the C.T17beta-GFP strain is cultivated to an optimum growth state, and is transferred according to proportion of 1:50 to a low nutrition medium containing organic pollutant with 0.05-1.0mM of concentration, the organic pollutants can be effectively degraded by continuous oscillating culture at the constant temperature of 27 DEG C. The invention has the beneficial effect that a comamonas testosterone modified strain is capable of simultaneously degrading a plurality of organic pollutants such as pesticide and the like. According to the GFP fluorescent effect, the method of the invention carries out fluorescence tracing in the process of degrading pollutants.
Description
Technical field
The invention belongs to microbial technology field; Relate to a kind of Comamonas testosteroni C.T17 β-GFP that utilizes the dna homolog recombinant technique to make up a strain green fluorescent protein mark, use in this plant mutant high-efficiency degradation environment multiple organic pollution and carry out fluorescence and follow the trail of.
Background technology
Comamonas testosteroni (Comamonas testosteroni; C.T) be a kind of in the Comamonas; Can be with steroid materials such as stosterones as the sole carbon source and the energy; Can also degrade xenobiotic, medicine, the agrochemical of non-steroid, the complicated metabolic pathway of the oxidation steroid nucleus through comprising many enzymes, this type of catapepsis substrate.
Green fluorescent protein (GFP) is the people such as to repair by the following village of Woods Hole marine biology research institute to find in jellyfish in 1962.The protein that its gene produced under the light of blue wavelength region excites, can send green fluorescent.This discovery has obtained Nobel chemistry Prize in 2008.In recent years the green fluorescence egg is widely used in fields such as biological marker.
Along with scientific technological advance, the environmental pollution that polycyclic aromatic hydrocarbon and residues of pesticides cause is serious day by day, has brought significant damage for human lives's health.The pollution of administering these two types of materials has become the domestic and international focus of attention.
Mainly contain the physics method, chemical method and bioanalysis to polycyclic aromatic hydrocarbons contaminated with improvement method pesticide residual contamination at present.Physical method changes pollutant generation form and place, thoroughly the contaminated solution problem.Chemical method has seriously hindered chemical method and has administered the technological development of organic pollution pollution because cost is higher.Compare with chemical recovery technique with physics, bioremediation technology has that cost is low, efficient is high, non-secondary pollution, do not destroy the required characteristics such as soil environment of growth.Bioremediation technology mainly is absorption, conversion, removing or the degradation water of utilizing plant, microorganism or protozoan etc., the organic pollution in the soil.Comprise phytoremediation, microorganism reparation, animal reparation etc.The relative degradation efficiency of phytoremediation is low, fully decomposing organic pollutant; Though animal is repaired degraded fully, this method is high to environmental requirement, is difficult to realize high degradation rate.It is to utilize the metabolic activity of microorganism to be converted into material even the mineralising that is prone to degraded to organic pollution that microorganism is repaired, and can transform into nontoxic metabolite to harmful substance, have easy and simple to handle, advantage such as be easy to handle on the spot.Thereby the microorganism recovery technique is a kind of improvement method that has broad prospect of application.
Summary of the invention
The objective of the invention is: the method that multiple organic pollution in a kind of green fluorescent protein microorganisms marked efficient degradation environment is provided; It can carry out efficient degradation with pesticide residual contamination to polycyclic aromatic hydrocarbons contaminated, in degraded, can follow the tracks of indication to the luminous intensity of green fluorescent protein.
Method of the present invention is:
1, obtain vector plasmid pK-GFP-2 through being connected behind the double digestion to plasmid pGEM-GFP and carrier pK18, be transformed into Escherichia coli amplification back and extract plasmid, enzyme is cut and is obtained the GFP fragment; Immediately the GFP fragment amplification product that reclaims being cut pTOPO17 β-HSD-600-3 with enzyme is connected; Obtained the plasmid of reorganization, called after pTOPO17 β-600-GFP transforms in the C.T competent cell through electricity; Obtain transforming strain C.T17 β-GFP through two resistance screenings, have following characteristic:
(1) have the GFP gene in the genomic DNA, its sequence is the sequence shown in the SEQ ID NO:1 in the sequence table;
(2) have plasmid pK18 gene in the genomic DNA, its sequence is the sequence shown in the SEQ ID NO:2 in the sequence table;
(3) the GFP gene of genomic DNA be positioned at C.T key enzyme 17beta-Hydroxysteroid dehydrogenase/carbonyl reductase (the gene downstream of 17 β-HSD), its sequence is the sequence shown in the SEQ ID NO:3 in the sequence table;
(4) have kanamycins (kannmycin) and the two resistances of ampicillin (ampicillin).
Above-mentioned pGEM-GFP and pK18 are the commercialization plasmid, and for buying in market, pTOPO17 β-600-3 plasmid is to be loaded with to comprise the carrier of 17 β-HSD promoter at 600 interior base-pairs, for this laboratory makes up.
2, C.T17 β-GFP bacterium is cultured to the optimum growh state, it is transferred in the low nutrient medium that contains the 0.05-1.0mM concentration organic pollutant, continue 27 ℃ of constant-temperature shaking culture, make organic pollution decomposed effectively according to 1: 50 ratio.
This technical scheme mainly comprises the steps:
(1) cultivation of C.T17 β-GFP bacterium: be inoculated in the liquid LB culture medium by 1: 50, under 27 ℃ of constant temperatures, 180-200 revolution per second shaken cultivation 12h;
(2) cultured C.T17 β-GFP bacterium was inoculated in the low nutrient medium that contains various organic pollutions by 1: 50, under the 20-27 ℃ of constant temperature, 180-200 revolution per second shaken cultivation;
(3) respectively at 24h, 48h, 72h, 96h, the 120h 3ml that takes a sample adopts spectrophotometric instrumentation 0D595 value;
(4) with the sample 3000-5000 revolution per second in (3) centrifugal 5-10 minute, get supernatant is measured its organic pollution with high performance liquid chromatography (HPLC) residual volume.
Organic pollution in the step (2) comprises benzene, xylenes, and naphthalene, pesticide D-D V, the S-fenvalerate, the degraded concentration range is: 0.05-1.0mM.
The high performance liquid chromatography testing result shows, this method is to benzene, xylenes, and the degradation rate of naphthalene all is higher than 60%, and to DDV, the degradation rate of pesticide materials such as S-fenvalerate all is higher than 40%.
The invention has the beneficial effects as follows: the present invention utilizes a kind of Comamonas testosteroni transformation strain C.T17 β-GFP simultaneously multiple organic pollutions such as agricultural chemicals to be carried out degradation experiment; Experimental result shows that this method is to benzene; Xylenes; The degradation rate of naphthalene all is higher than 60%, and to DDV, the degradation rate of pesticide materials such as S-fenvalerate all is higher than 40%.Simultaneously, each is according to the fluorescence of GFP, and the present invention has carried out the fluorescence tracking in the process of degradation of contaminant, for the monitoring of degradation process lays the first stone.This method multiple organic pollution in the environment of can degrading; And efficient is higher, and cost is lower, non-secondary pollution, does not destroy the required soil environment of growth; Can carry out real-time fluorescence to degradation process simultaneously and follow the tracks of indication, be the better method of organic pollution in the degraded environment.
Description of drawings
Fig. 1 is pTOPO17 β-600-GFP building process.
Fig. 2 is an organic concentration when being the best, and reaction begins to 15h the detected fluorescent value of 136h.
Fig. 3 is an organic concentration when being the best, and reaction begins to the detected sample residual quantity of 136h high performance liquid chromatography.
The specific embodiment
Embodiment 1 C.T17 β-GFP bacterium is to the degradation experiment of light concentration organic pollutant
(1) double digestion of plasmid pGEM-GFP and carrier pK18
Reaction system is following:
Become partial volume
Each 1 μ l of EcoR I/Bamh I
pGEM-GFP/pK18 25μl
10* enzyme cutting buffering liquid 3 μ l
37℃，5h
Obtain the GFP fragment, carry out agarose gel electrophoresis (1%) and identify, reclaim the purpose fragment.
(2) purpose fragment GFP and carrier pK18's is connected
With the GFP fragment double digestion pcr amplification product that reclaims immediately with being connected of double digestion pK18, reaction system is following:
Become partial volume
Double digestion pK18 4 μ l
GFP fragment 1 μ l
T4 ligase 1 μ l
10*T4 enzyme buffer liquid 1 μ l
ATP(5mM) 1μl
ddH
2O 2μl
16℃，20h
Obtain plasmid pK-GFP-2, be transformed into the Escherichia coli amplification.
(3) enzyme of plasmid pKGFP and pTOPO17 β-600-3 is cut
The pKGFP that Escherichia coli amplifications is obtained and pTOPO17 β-600-3 carry out enzyme respectively and cut, and reaction system is following:
The pKGFP enzyme is cut:
Become partial volume
Each 1 μ l of BamH I/Pst II
pKGFP 25μl
10* enzyme cutting buffering liquid 3 μ l
37℃，5h
PTOPO17 β-600-3 enzyme is cut:
Become partial volume
Each 1 μ l of BamH I/Pst I/Hind III
pTOPO17β-600-3 25μl
10* enzyme cutting buffering liquid 3 μ l
37℃，5h
(4) purpose fragment GFP and plasmid pTOPO17 β-600-3's is connected
The GFP fragment amplification product that reclaims is cut pTOPO17 β-600-3 with enzyme immediately be connected, reaction system is following:
Become partial volume
Enzyme is cut pTOPO17 β-600-3 4 μ l
GFP fragment 1 μ l
T4 ligase 1 μ l
10*T4 enzyme buffer liquid 1 μ l
ATP(5mM) 1μl
ddH
2O 2μl
16℃，20h
Obtain plasmid pTOPO17 β-600-GFP; It has kalamycin resistance; Can in Escherichia coli, duplicate; And can not in the C.T bacterium, duplicate, plasmid pTOPO17 β-600-GFP is advanced in the C.T competent cell through electric conversion, obtain mutant strain C.T17 β-GFP through two resistance screenings (kannmycin+ampicillin).
(5), C.T17 β-GFP bacterium is to the degradation experiment of light concentration organic pollutant
(1) cultivation of C.T17 β-GFP bacterium: be inoculated in the liquid LB culture medium by 1: 50, under 27 ℃ of constant temperatures, 180-200 revolution per second shaken cultivation is spent the night;
(2) cultured C.T17 β-GFP bacterium was inoculated in the benzene that concentration is 0.05mM by 1: 50, xylenes, naphthalene, DDV, in the low nutrient medium of materials such as S-fenvalerate, under 27 ℃ of constant temperatures, 180-200 revolution per second shaken cultivation;
(3) respectively at 24h, 48h, 72h, 96h, 120h, the 136h 3ml that takes a sample adopts spectrophotometric instrumentation OD595 value;
(4) with the sample 3000-5000 revolution per second in (3) centrifugal 5-10 minute, get supernatant is measured its organic pollution with high performance liquid chromatography (HPLC) residual volume.
High performance liquid chromatography testing result such as following table:
Table 1. C.T17 β-GFP is to the degraded of light concentration organic pollutant
(1) double digestion of plasmid pGEM-GFP and carrier pK18
Reaction system is following:
Become partial volume
Each 1 μ l of EcoR I/Bamh I
pGEM-GFP/pK18 25μl
10* enzyme cutting buffering liquid 3 μ l
37℃，5h
Obtain the GFP fragment, carry out agarose gel electrophoresis (1%) and identify, reclaim the purpose fragment.
(2) purpose fragment GFP and carrier pK18's is connected
With the GFP fragment double digestion pcr amplification product that reclaims immediately with being connected of double digestion pK18, reaction system is following:
Become partial volume
Double digestion pK18 4 μ l
GFP fragment 1 μ l
T4 ligase 1 μ l
10*T4 enzyme buffer liquid 1 μ l
ATP(5mM) 1μl
ddH
2O 2μl
16℃，20h
Obtain plasmid pK-GFP-2, be transformed into the Escherichia coli amplification.
(3) enzyme of plasmid pKGFP and pTOPO17 β-600-3 is cut
The pKGFP that Escherichia coli amplifications is obtained and pTOPO17 β-600-3 carry out enzyme respectively and cut, and reaction system is following:
The pKGFP enzyme is cut:
Become partial volume
Each 1 μ l of BamH I/Pst II
pKGFP 25μl
10* enzyme cutting buffering liquid 3 μ l
37℃，5h
PTOPO17 β-600-3 enzyme is cut:
Become partial volume
Each 1 μ l of BamH I/Pst I/Hind III
pTOPO17β-600-3 25μl
10* enzyme cutting buffering liquid 3 μ l
37℃，5h
(4) purpose fragment GFP and plasmid pTOPO17 β-600-3's is connected
The GFP fragment amplification product that reclaims is cut pTOPO17 β-600-3 with enzyme immediately be connected, reaction system is following:
Become partial volume
Enzyme is cut pTOPO17 β-600-3 4 μ l
GFP fragment 1 μ l
T4 ligase 1 μ l
10*T4 enzyme buffer liquid 1 μ l
ATP(5mM) 1μl
ddH
2O 2μl
16℃，20h
Obtain plasmid pTOPO17 β-600-GFP; It has kalamycin resistance; Can in Escherichia coli, duplicate; And can not in the C.T bacterium, duplicate, plasmid pTOPO17 β-600-GFP is advanced in the C.T competent cell through electric conversion, obtain mutant strain C.T17 β-GFP through two resistance screenings (kannmycin+ampicillin).
(5), C.T17 β-GFP bacterium is to DDV, the degradation experiment of pesticide materials such as S-fenvalerate
(1) cultivation of C.T17 β-GFP bacterium: be inoculated in the liquid LB culture medium by 1: 50, under 27 ℃ of constant temperatures, 180-200 revolution per second shaken cultivation is spent the night;
(2) cultured C.T17 β-GFP bacterium was inoculated in the benzene that concentration is 0.1mM by 1: 50, xylenes, naphthalene, DDV, in the low nutrient medium of materials such as S-fenvalerate, under 27 ℃ of constant temperatures, 180-200 revolution per second shaken cultivation;
(3) respectively at 24h, 48h, 72h, 96h, 120h, the 136h 3ml that takes a sample adopts spectrophotometric instrumentation OD595 value;
(4) with the sample 3000-5000 revolution per second in (3) centrifugal 5-10 minute, get supernatant is measured its organic pollution with high performance liquid chromatography (HPLC) residual volume.
The high performance liquid chromatography testing result sees the following form:
Table 2. C.T17 β-GFP bacterium is to the degraded of higher concentration organic pollutant
Embodiment 3 C.T17 β-GFP bacterium is to the degradation experiment of high concentration organic contaminant
(1) double digestion of plasmid pGEM-GFP and carrier pK18
Reaction system is following:
Become partial volume
Each 1 μ l of EcoR I/Bamh I
pGEM-GFP/pK18 25μl
10* enzyme cutting buffering liquid 3 μ l
37℃，5h
Obtain the GFP fragment, carry out agarose gel electrophoresis (1%) and identify, reclaim the purpose fragment.
(2) purpose fragment GFP and carrier pK18's is connected
With the GFP fragment double digestion pcr amplification product that reclaims immediately with being connected of double digestion pK18, reaction system is following:
Become partial volume
Double digestion pK18 4 μ l
GFP fragment 1 μ l
T4 ligase 1 μ l
10*T4 enzyme buffer liquid 1 μ l
ATP(5mM) 1μl
ddH
2O 2μl
16℃，20h
Obtain plasmid pK-GFP-2, be transformed into the Escherichia coli amplification.
(3) enzyme of plasmid pKGFP and pTOPO17 β-600-3 is cut
The pKGFP that Escherichia coli amplifications is obtained and pTOPO17 β-600-3 carry out enzyme respectively and cut, and reaction system is following: the pKGFP enzyme is cut:
Become partial volume
Each 1 μ l of BamH I/Pst II
pKGFP 25μl
10* enzyme cutting buffering liquid 3 μ l
37℃，5h
PTOPO17 β-600-3 enzyme is cut:
Become partial volume
Each 1 μ l of BamH I/Pst I/Hind III
pTOPO17β-600-3 25μl
10* enzyme cutting buffering liquid 3 μ l
37℃，5h
(4) purpose fragment GFP and plasmid pTOPO17 β-600-3's is connected
The GFP fragment amplification product that reclaims is cut pTOPO17 β-600-3 with enzyme immediately be connected, reaction system is following:
Become partial volume
Enzyme is cut pTOPO17 β-600-3 4 μ l
GFP fragment 1 μ l
T4 ligase 1 μ l
10*T4 enzyme buffer liquid 1 μ l
ATP(5mM) 1μl
ddH
2O 2μl
16℃，20h
Obtain plasmid pTOPO17 β-600-GFP; It has kalamycin resistance; Can in Escherichia coli, duplicate; And can not in the C.T bacterium, duplicate, plasmid pTOPO17 β-600-GFP is advanced in the C.T competent cell through electric conversion, obtain mutant strain C.T17 β-GFP through two resistance screenings (kannmycin+ampicillin).
(5), C.T17 β-GFP bacterium is to DDV, the degradation experiment of pesticide materials such as S-fenvalerate
(1) cultivation of C.T17 β-GFP bacterium: be inoculated in the liquid LB culture medium by 1: 50, under 27 ℃ of constant temperatures, 180-200 revolution per second shaken cultivation is spent the night;
(2) cultured C.T17 β-GFP bacterium was inoculated in the benzene that concentration is 1.0mM by 1: 50, xylenes, naphthalene, DDV, in the low nutrient medium of materials such as S-fenvalerate, under 27 ℃ of constant temperatures, 180-200 revolution per second shaken cultivation;
(3) respectively at 24h, 48h, 72h, 96h, 120h, the 136h 3ml that takes a sample adopts spectrophotometric instrumentation OD595 value;
(4) with the sample 3000-5000 revolution per second in (3) centrifugal 5-10 minute, get supernatant is measured its organic pollution with high performance liquid chromatography (HPLC) residual volume.
The high performance liquid chromatography testing result sees the following form:
Table 3. C.T17 β-GFP bacterium is to the degraded of high concentration organic contaminant
Embodiment 4 C.T17 β-GFP bacterium is to the fluorescence real-time tracing monitoring of organic pollutant degradation process
(1) double digestion of plasmid pGEM-GFP and carrier pK18
Reaction system is following:
Become partial volume
Each 1 μ l of EcoR I/Bamh I
pGEM-GFP/pK18 25μl
10* enzyme cutting buffering liquid 3 μ l
37℃，5h
Obtain the GFP fragment, carry out agarose gel electrophoresis (1%) and identify, reclaim the purpose fragment.
(2) purpose fragment GFP and carrier pK18's is connected
With the GFP fragment double digestion pcr amplification product that reclaims immediately with being connected of double digestion pK18, reaction system is following:
Become partial volume
Double digestion pK18 4 μ l
GFP fragment 1 μ l
T4 ligase 1 μ l
10*T4 enzyme buffer liquid 1 μ l
ATP(5mM) 1μl
ddH
2O 2μl
16℃，20h
Obtain plasmid pK-GFP-2, be transformed into the Escherichia coli amplification.
(3) enzyme of plasmid pKGFP and pTOPO17 β-600-3 is cut
The pKGFP that Escherichia coli amplifications is obtained and pTOPO17 β-600-3 carry out enzyme respectively and cut, and reaction system is following:
The pKGFP enzyme is cut:
Become partial volume
Each 1 μ l of BamH I/Pst II
pKGFP 25μl
10* enzyme cutting buffering liquid 3 μ l
37℃，5h
PTOPO17 β-600-3 enzyme is cut:
Become partial volume
Each 1 μ l of BamH I/Pst I/Hind III
pTOPO17β-600-3 25μl
10* enzyme cutting buffering liquid 3 μ l
37℃，5h
(4) purpose fragment GFP and plasmid pTOPO17 β-600-3's is connected
The GFP fragment amplification product that reclaims is cut pTOPO17 β-600-3 with enzyme immediately be connected, reaction system is following:
Become partial volume
Enzyme is cut pTOPO17 β-600-3 4 μ l
GFP fragment 1 μ l
T4 ligase 1 μ l
10*T4 enzyme buffer liquid 1 μ l
ATP(5mM) 1μl
ddH
2O 2μl
16℃，20h
Obtain plasmid pTOPO17 β-600-GFP; It has kalamycin resistance; Can in Escherichia coli, duplicate; And can not in the C.T bacterium, duplicate, plasmid pTOPO17 β-600-GFP is transformed in the C.T competent cell through electricity, obtain mutant strain C.T17 β-GFP through two resistance screenings (kannmycin+ampicillin).
(5), C.T17 β-GFP bacterium is to DDV, the degradation experiment of pesticide materials such as S-fenvalerate
(1) cultivation of C.T17 β-GFP bacterium: be inoculated in the liquid LB culture medium by 1: 50, under 27 ℃ of constant temperatures, 180-200 revolution per second shaken cultivation is spent the night;
(2) cultured C.T17 β-GFP bacterium was inoculated in the DDV that concentration is respectively optium concentration 0.1mM by 1: 50, in the low nutrient medium of materials such as S-fenvalerate, under 27 ℃ of constant temperatures, 180-200 revolution per second shaken cultivation;
(3) respectively at 24h, 48h, 72h, 96h, the 120h 3ml that takes a sample adopts spectrophotometric instrumentation OD595 value;
(4) with the sample 3000-5000 revolution per second in (3) centrifugal 5-10 minute, get supernatant is measured its organic pollution with high performance liquid chromatography (HPLC) residual volume.
The high performance liquid chromatography testing result shows that this method is 57% to the degradation rate of DDV, is 43% to the degradation rate of S-fenvalerate.
(6) C.T17 β-GFP bacterium is to the fluorescence real-time tracing monitoring of organic pollutant degradation process
(1) cultivation of C.T17 β-GFP bacterium: be inoculated in the liquid LB culture medium by 1: 50, under 27 ℃ of constant temperatures, 180-200 revolution per second shaken cultivation is spent the night;
(2) cultured C.T17 β-GFP bacterium was inoculated in benzene, xylenes, naphthalene, the DDV that concentration is respectively optium concentration 0.1mM by 1: 50, in the low nutrient medium of materials such as S-fenvalerate, under 27 ℃ of constant temperatures, 180-200 revolution per second shaken cultivation;
(3) respectively at 24h, 48h, 72h, 96h, 120h, the 136h 3ml that takes a sample measures fluorescent value, and testing conditions: excitation wavelength is 485nm, and emission wavelength is 525nm.
Claims (2)
1. the method for multiple organic pollution in the green fluorescent protein microorganisms marked efficient degradation environment, its method is:
A, obtain vector plasmid pK-GFP-2 through being connected behind the double digestion to plasmid pGEM-GFP and carrier pK18; Be transformed into Escherichia coli amplification back and extract plasmid, enzyme is cut and is obtained the GFP fragment, immediately the GFP fragment amplification product that reclaims is cut pTOPO17 β-HSD-600-3 with enzyme and is connected; Obtained the plasmid of reorganization; Called after pTOPO17 β-600-GFP transforms in the C.T competent cell through electricity, obtains transforming strain C.T17 β-GFP through two resistance screenings;
Have the GFP gene in a genomic DNA, its sequence is the sequence shown in the SEQ ID NO:1 in the sequence table;
Have plasmid pK18 gene in the b genomic DNA, its sequence is the sequence shown in the SEQ ID NO:2 in the sequence table;
The GFP gene of c genomic DNA is positioned at C.T key enzyme 17beta-Hydroxysteroid dehydrogenase/carbonyl reductase 17 β-HSD gene downstream, and its sequence is the sequence shown in the SEQ ID NO:3 in the sequence table;
D has kanamycins and the two resistances of ampicillin;
B, C.T17 β-GFP bacterium is cultured to the optimum growh state, it is transferred in the low nutrient medium that contains the 0.05-1.0mM concentration organic pollutant, continue 27 ℃ of constant-temperature shaking culture, make organic pollution decomposed effectively according to 1: 50 ratio.
2. the method for multiple organic pollution in a kind of green fluorescent protein microorganisms marked efficient degradation environment according to claim 1, its method is:
The cultivation of A, C.T17 β-GFP bacterium: be inoculated in the liquid LB culture medium by 1: 50, under 27 ℃ of constant temperatures, 180-200 revolution per second shaken cultivation 12h;
B, cultured C.T17 β-GFP bacterium was inoculated in the low nutrient medium that contains various organic pollutions by 1: 50, under the 20-27 ℃ of constant temperature, 180-200 revolution per second shaken cultivation;
C, respectively at 24h, 48h, 72h, 96h, the 120h 3ml that takes a sample adopts spectrophotometric instrumentation OD595 value;
D, with the sample 3000-5000 revolution per second among the step C centrifugal 5-10 minute, get the residual volume of supernatant with its organic pollution of high effective liquid chromatography for measuring.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1055732A1 (en) * | 1998-12-18 | 2000-11-29 | Kaneka Corporation | Process for producing (r)-2-hydroxy-1-phenoxypropane derivative |
| CN101539524A (en) * | 2009-03-06 | 2009-09-23 | 于源华 | Fluorescence detection card and fluorescence detection method for organic contaminants in food and environment |
-
2010
- 2010-08-12 CN CN2010102512792A patent/CN102371042A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1055732A1 (en) * | 1998-12-18 | 2000-11-29 | Kaneka Corporation | Process for producing (r)-2-hydroxy-1-phenoxypropane derivative |
| CN101539524A (en) * | 2009-03-06 | 2009-09-23 | 于源华 | Fluorescence detection card and fluorescence detection method for organic contaminants in food and environment |
Non-Patent Citations (3)
| Title |
|---|
| GUANGMING XIONG等: "3α-Hydroxysteroid dehydrogenase/carbonyl reductase as a tool for isolation and characterization of a new marine steroid degrading bacterial strain", 《CHEMICO-BIOLOGICAL INTERACTIONS》 * |
| 于源华等: "生物荧光方法快速测定环境水体中的甾体类激素", 《分析化学》 * |
| 陈潇滢: "《硕士学位论文》", 28 February 2010 * |
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