CN103232999B - Degradative plasmid containing functional gene capable of degradating benz(a)anthracene - Google Patents
Degradative plasmid containing functional gene capable of degradating benz(a)anthracene Download PDFInfo
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Abstract
The invention provides a degradative plasmid containing a functional gene capable of degrading benz(a)anthracene. The degradative plasmid containing the functional gene capable of degrading benz(a)anthracene is obtained through extracting and purifying a strain solution of a strain-Stenotrophomonas acidaminiphila. The degradative plasmid is prepared according to the following steps: inoculating the strain-Stenotrophomonas acidaminiphila into a glass bottle added with benz(a)anthracene, a basic culture medium, a trace metal liquid and a vitamin C solution, culturing for 20 days so as to obtain the strain solution, and extracting and purifying the strain solution so as to obtain the degradative plasmid containing the functional gene capable of degrading benz(a)anthracene. The obtained degradative plasmid containing the functional gene capable of degrading benz(a)anthracene can be used for forming the strain with a degrading function on the benz(a)anthracene, and has a biological degradation effect on polycyclic aromatic hydrocarbon pollutants such as a representative-benz(a)anthracene and application potential for the soil and water body pollution of the polycyclic aromatic hydrocarbon-benz(a)anthracene.
Description
Technical field
The invention belongs to microbiology and technical field of molecular biology, be specifically related to a kind of dissimilation plasmid containing degraded benzo [a] anthracene functional gene.
Background technology
Polycyclic aromatic hydrocarbons (Polycyclic Aromatic Hydrocarbons, PAHs) is the toxic organic pollutant that a class contains two or more phenyl ring, has strong carcinogenesis, teratogenesis and mutagenesis.PAHs causes high risks by the transmission effect of biological accumulation and food chain to ecotope and HUMAN HEALTH, has caused the great attention of various countries environmentalist.Mainly contain Physical, chemical method and biological process to the Treatment process of Polycyclic Aromatic Hydrocarbonat Existing in Environment, wherein biological process has simple to operate, the advantage such as working cost is low, non-secondary pollution.In general, along with the increase of polycyclic aromatic hydrocarbons phenyl ring quantity, its degradation rate reduces.Low-molecular-weight polycyclic aromatic hydrocarbons can comparatively fast be degraded in the environment, the time existed in the environment is shorter, and the polycyclic aromatic hydrocarbons of high molecular, such as pyrene, fluoranthene, benzo [a] pyrene, benzo [a] anthracene, indeno [1,2,3-cd] pyrene, benzo [b] fluoranthene and benzo [k] fluoranthene etc. be then difficult to degraded, longer-term is present in environment.Therefore, structure polycyclic aromatic hydrocarbons efficient degrading bacteria carrys out enhancing degradation has become the polycyclic aromatic hydrocarbons contaminated a kind of important method of removal.
Degrading polycyclic aromatic hydrocarbons plasmid is can the small molecule DNA of double-stranded circular of self-replacation in cell, and the metabolic enzyme of aromatics of degrading is normally by dissimilation plasmid genes encoding.Therefore, degrading polycyclic aromatic hydrocarbons plasmid carries the degrading genes information of part, be connected in host bacteria by transformation and form recombinant bacterium, thus make recombinant bacterium show the performance of corresponding degrading polycyclic aromatic hydrocarbons.From polycyclic aromatic hydrocarbon-degrading bacteria strain, extraction and purification obtains degrading polycyclic aromatic hydrocarbons plasmid and all has great importance for building new polycyclic aromatic hydrocarbons efficient degrading bacteria and improving the degradation property of polycyclic aromatic hydrocarbons.But, lack the research report in degrading polycyclic aromatic hydrocarbons plasmid both at home and abroad.
Summary of the invention
The invention provides a kind of dissimilation plasmid containing degraded benzo [a] anthracene functional gene.The described dissimilation plasmid containing degraded benzo [a] anthracene functional gene is obtained through extraction and purification by the bacterium liquid of bacterial strain Stenotrophomonas acidaminiphila.The bacterial strain Stenotrophomonas acidaminiphila that extraction and purification contains the dissimilation plasmid of degraded benzo [a] anthracene functional gene used is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 23rd, 2010, this is centrally located at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, and culture presevation number is CGMCC No.3683.The concrete scheme that extraction and purification contains the dissimilation plasmid of degraded benzo [a] anthracene functional gene from bacterial strain Stenotrophomonas acidaminiphila is:
(1) to volume be 500mL Erlenmeyer flask in add 400mL basic medium, 1.0mL trace metal liquid, 0.1mL vitamin c solution, shake up rear for subsequent use as liquid nutrient medium; Wherein the consisting of of basic medium: NH
4nO
3: 1.7gL
-1, KH
2pO
4: 1.0gL
-1, K.
2hPO
4: 1.0gL
-1, MgCl
2: 0.30gL
-1, CaCl
22H
2o:0.15gL
-1; Consisting of of trace metal liquid: CoCl
26H
2o:55mgL
-1, CuCl
2: 0.35mgL
-1, H
3bO
3: 11.0mgL
-1, MnCl
24H
2o:45mgL
-1, Na
2moO
42H
2o:5.5mgL
-1, NiCl
22H
2o:4.5mgL
-1, ZnCl
2: 4.5mgL
-1;
(2) be in the Erlenmeyer flask of 100mL, add benzo [a] the anthracene acetone soln that 6.0mL concentration is 1g/L to volume in Bechtop, for Erlenmeyer flask opening is placed 2 days by removing acetone;
(3) in the Erlenmeyer flask in step (2), add the liquid nutrient medium that 60mL prepares by step (1);
(4) picking bacterial strain Stenotrophomonas acidaminiphila in Bechtop, is seeded in the Erlenmeyer flask in step (3), temperature be 25 ~ 30 DEG C, rotating speed be in the vibrator of 100r/min cultivate 20 ~ 22 days;
(5) the bacterium liquid in step (4) being transferred to volume is in the centrifuge tube of 15mL, centrifugal 10min under 10000r/min condition, abandoning supernatant;
(6) in the centrifuge tube in step (5), add 10mL pH value be 9.0 concentration is the NaH of 0.08M
2pO
4damping fluid, carry out ultra-sonic oscillation 15min again, centrifugal 10min under 10000r/min condition after vortex vibration 5min, abandoning supernatant isolates thalline;
(7) in the centrifuge tube by step (6), add 7mL solution A, then mix; Consisting of of solution A: 50mL0.45M trishydroxymethylaminomethane-HCl, 20mL0.25M EDTA, 20mL0.9M KCl, 10mL percentage concentration is the hexadecyl Trimethylamine 99 of 2.0%, pH8.2; Then 2.2mgN-acetylmuramide glycanohydrla is added, vibrate under 0 ~ 4 DEG C of condition after 10min and add the ethoxylated alkyl sulfate solution that 1.5mL concentration is 50g/L, at 55 DEG C of Water Under bath vibration 1h after vortex vibration 5min, centrifugal 10min under 10000r/min condition, collects supernatant liquor;
(8) in the supernatant liquor obtained by step (7), add the Potassium ethanoate and 0.5mL Virahol that 1mL concentration is 5.5M, vibrate 15min under 0 ~ 4 DEG C of condition, centrifugal 10min under 10000r/min condition, collects supernatant liquor;
(9) join on centrifugal purification post by the supernatant liquor obtained by step (8), centrifugal purification post is put into centrifuge tube, centrifugal 10min under 10000r/min condition, removes the liquid in centrifuge tube; Repeat this process, until all supernatant liquors obtained by step (8) are all through centrifugal purification column purification;
(10) in the centrifugal purification post after step (9) process, 0.5mL solution B is added, consisting of of solution B: 5mL4.5M Guanidinium hydrochloride, 10mL3.5M Potassium ethanoate and 85mL dehydrated alcohol, pH7.0; Vibrate 15min under 0 ~ 4 DEG C of condition, and centrifugal 5min under 10000r/min condition, removes the liquid in centrifuge tube;
(11) in the centrifugal purification post after step (10) process, 0.6mL solution C is added, consisting of of solution C: 40mL10mM trishydroxymethylaminomethane-HCl, 30mL3.0M NaCl, 30mL1.5mM EDTA, pH8.5; Vibrate 20min under 55 DEG C of conditions, centrifugal 5min under 10000r/min condition; Liquid in centrifuge tube is rejoined in this centrifugal purification post, in 55 DEG C of water-baths, place 10min, centrifugal 5min under 10000r/min condition; In centrifuge tube, liquid is the dissimilation plasmid solution containing degraded benzo [a] anthracene functional gene, saves backup at-20 DEG C.
The dissimilation plasmid containing degraded benzo [a] anthracene functional gene that the present invention obtains can build bacterial strain benzo [a] anthracene to degradation function, raising take benzo [a] anthracene as the biological degradation effect of the polycyclic aromatic hydrocarbon pollutant of representative, has application potential for polycyclic aromatic hydrocarbons benzo [a] anthracene contaminated soil and water body.
Embodiment
The present invention is further illustrated below in conjunction with example.
Material:
(1) bacterial strain Stenotrophomonas acidaminiphila, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 23rd, 2010, this is centrally located at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, and culture presevation number is CGMCC No.3683.
(2) e. coli bl21 competent cell (Tianjin Bo Meike Bioisystech Co., Ltd).
(3) benzo [a] anthracene (purity >=99%, Sigma Aldrich).
(4) acetone, N-acetylmuramide lycanohydrlase, Virahol.
(5) NaH of pH value to be 9.0 concentration be 0.08M
2pO
4damping fluid.
(6) concentration is the ethoxylated alkyl sulfate solution of 50g/L.
(7) concentration is the liquor kalii acetici of 5.5M.
(8) solution A 100mL, it consists of: 50mL0.45M trishydroxymethylaminomethane-HCl, and 20mL0.25MEDTA, 20mL0.9M KCl, 10mL percentage concentration is the hexadecyl Trimethylamine 99 of 2.0%, pH8.2.
(9 solution B 100mL, it consists of: 5mL4.5M Guanidinium hydrochloride, 10mL3.5M Potassium ethanoate and 85mL dehydrated alcohol, pH7.0.
(10) solution C 100mL, it consists of: 40mL10mM trishydroxymethylaminomethane-HCl, 30mL3.0MNaCl, 30mL1.5mM EDTA, pH8.5.
(11) basic medium 1L, the concentration of its composition and each component is: NH
4nO
3: 1.7gL
-1, KH
2pO
4: 1.0gL
-1, K.
2hPO
4: 1.0gL
-1, MgCl
2: 0.30gL
-1, CaCl
22H
2o:0.15gL
-1.
(12) trace metal liquid 1L, the concentration of its composition and each component is: CoCl
26H
2o:55mgL
-1, CuCl
2: 0.35mgL
-1, H
3bO
3: 11.0mgL
-1, MnCl
24H
2o:45mgL
-1, Na
2moO
42H
2o:5.5mgL
-1, NiCl
22H
2o:4.5mgL
-1, ZnCl
2: 4.5mgL
-1.
Embodiment
The bacterial strain Stenotrophomonas acidaminiphila at China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved in the interior inoculation of the vial adding benzo [a] anthracene and basic medium, trace metal liquid and vitamin c solution, then by culturing bottle culture temperature be 25 ~ 28 DEG C, rotating speed is cultivate 20d in the vibrator of 100r/min, obtains the bacterium liquid after amplification cultivation.By bacterium liquid centrifugal 10min under 10000r/min condition, in the thalline be separated, to add 10mL pH value be 9.0 concentration is the NaH of 0.08M
2pO
4damping fluid, carry out ultra-sonic oscillation 15min again after vortex vibration 5min, centrifugal 10min under 10000r/min condition, adds 7mL solution A and mixes after abandoning supernatant.Then 2.2mg N-acetylmuramide lycanohydrlase is added, vibrate under 0 ~ 4 DEG C of condition after 10min and add the ethoxylated alkyl sulfate solution that 1.5mL concentration is 50g/L, at 55 DEG C of Water Under bath vibration 1h after vortex vibration 5min, centrifugal 10min under 10000r/min condition, collects supernatant liquor.Then in the supernatant liquor collected, add the Potassium ethanoate that 1mL concentration is 5.5M and 0.5mL Virahol, vibrate 15min under 0 ~ 4 DEG C of condition, centrifugal 10min under 10000r/min condition, collects supernatant liquor.Joined by the supernatant liquor collected in centrifugal purification post, then centrifugal 10min under 10000r/min condition, removes the liquid in centrifuge tube.Repeat this process, until all supernatant liquors are all through centrifugal purification column purification.In centrifugal purification post, add 0.5mL solution B, centrifugal 5min under 10000r/min condition, removes the liquid in centrifuge tube.Then in centrifugal purification post, add 0.6mL solution C, vibrate 20min under 55 DEG C of conditions, centrifugal 5min under 10000r/min condition.Liquid in centrifuge tube is rejoined in this centrifugal purification post, in 55 DEG C of water-baths, place 10min, centrifugal 5min under 10000r/min condition.In centrifuge tube, liquid is the dissimilation plasmid solution containing degraded benzo [a] anthracene functional gene, saves backup at-20 DEG C.
Dissimilation plasmid containing degraded benzo [a] anthracene functional gene is transferred in e. coli bl21 by the method using electricity to transform, the recombinant bacterium obtained can be grow in the minimal medium of sole carbon source with benzo [a] anthracene, and to benzo [a] anthracene, there is degradation function, use the recombinant bacterium obtained to carry out Degrading experiment to benzo [a] the anthracene solution that starting point concentration is 0.11mg/L, after result shows 7 days, the clearance of benzo [a] anthracene reaches 92.7%.
Claims (1)
1. the dissimilation plasmid containing degraded benzo [a] anthracene functional gene, it is characterized in that, this dissimilation plasmid is obtained through extraction and purification by the bacterium liquid of bacterial strain Stenotrophomonas acidaminiphila, the culture presevation number of bacterial strain Stenotrophomonas acidaminiphila is CGMCC No.3683, wherein, the concrete scheme that extraction and purification contains the dissimilation plasmid of degraded benzo [a] anthracene functional gene from bacterial strain Stenotrophomonas acidaminiphila is:
(1) to volume be 500mL Erlenmeyer flask in add 400mL basic medium, 1.0mL trace metal liquid, 0.1mL vitamin c solution, shake up rear for subsequent use as liquid nutrient medium; Wherein the consisting of of basic medium: NH
4nO
3: 1.7gL
-1, KH
2pO
4: 1.0gL
-1, K
2hPO
4: 1.0gL
-1, MgCl
2: 0.30gL
-1, CaCl
22H
2o:0.15gL
-1; Consisting of of trace metal liquid: CoCl
26H
2o:55mgL
-1, CuCl
2: 0.35mgL
-1, H
3bO
3: 11.0mgL
-1, MnCl
24H
2o:45mgL
-1, Na
2moO
42H
2o:5.5mgL
-1, NiCl
22H
2o:4.5mgL
-1, ZnCl
2: 4.5mgL
-1;
(2) be in the Erlenmeyer flask of 100mL, add benzo [a] the anthracene acetone soln that 6.0mL concentration is 1g/L to volume in Bechtop, for Erlenmeyer flask opening is placed 2 days by removing acetone;
(3) in the Erlenmeyer flask in step (2), add the liquid nutrient medium that 60mL prepares by step (1);
(4) picking bacterial strain Stenotrophomonas acidaminiphila in Bechtop, is seeded in the Erlenmeyer flask in step (3), temperature be 25 ~ 30 DEG C, rotating speed be in the vibrator of 100r/min cultivate 20 ~ 22 days;
(5) the bacterium liquid in step (4) being transferred to volume is in the centrifuge tube of 15mL, centrifugal 10min under 10000r/min condition, abandoning supernatant;
(6) in the centrifuge tube in step (5), add 10mL pH value be 9.0 concentration is the NaH of 0.08M
2pO
4damping fluid, carry out ultra-sonic oscillation 15min again, centrifugal 10min under 10000r/min condition after vortex vibration 5min, abandoning supernatant isolates thalline;
(7) in the centrifuge tube by step (6), add 7mL solution A, then mix; Consisting of of solution A: 50mL0.45M trishydroxymethylaminomethane-HCl, 20mL0.25M EDTA, 20mL0.9M KCl, 10mL percentage concentration is the hexadecyl Trimethylamine 99 of 2.0%, pH8.2; Then 2.2mg N-acetylmuramide lycanohydrlase is added, vibrate under 0 ~ 4 DEG C of condition after 10min and add the ethoxylated alkyl sulfate solution that 1.5mL concentration is 50g/L, at 55 DEG C of Water Under bath vibration 1h after vortex vibration 5min, centrifugal 10min under 10000r/min condition, collects supernatant liquor;
(8) in the supernatant liquor obtained by step (7), add the Potassium ethanoate and 0.5mL Virahol that 1mL concentration is 5.5M, vibrate 15min under 0 ~ 4 DEG C of condition, centrifugal 10min under 10000r/min condition, collects supernatant liquor;
(9) join on centrifugal purification post by the supernatant liquor obtained by step (8), centrifugal purification post is put into centrifuge tube, centrifugal 10min under 10000r/min condition, removes the liquid in centrifuge tube; Repeat this process, until all supernatant liquors obtained by step (8) are all through centrifugal purification column purification;
(10) in the centrifugal purification post after step (9) process, 0.5mL solution B is added, consisting of of solution B: 5mL4.5M Guanidinium hydrochloride, 10mL3.5M Potassium ethanoate and 85mL dehydrated alcohol, pH7.0; Vibrate 15min under 0 ~ 4 DEG C of condition, and centrifugal 5min under 10000r/min condition, removes the liquid in centrifuge tube;
(11) in the centrifugal purification post after step (10) process, 0.6mL solution C is added, consisting of of solution C: 40mL10mM trishydroxymethylaminomethane-HCl, 30mL3.0M NaCl, 30mL1.5mM EDTA, pH8.5; Vibrate 20min under 55 DEG C of conditions, centrifugal 5min under 10000r/min condition; Liquid in centrifuge tube is rejoined in this centrifugal purification post, in 55 DEG C of water-baths, place 10min, centrifugal 5min under 10000r/min condition; In centrifuge tube, liquid is the dissimilation plasmid solution containing degraded benzo [a] anthracene functional gene, saves backup at-20 DEG C.
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Citations (3)
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| CN101514224A (en) * | 2008-02-20 | 2009-08-26 | 北京师范大学 | Method for extracting and purifying zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon |
| CN101974443A (en) * | 2010-03-02 | 2011-02-16 | 北京师范大学 | Benzo[a]pyrene-degrading strain and application thereof |
| CN102533578A (en) * | 2010-12-10 | 2012-07-04 | 中国农业大学 | Microorganism bacterium agent for degrading polycyclic aromatic hydrocarbon |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101514224A (en) * | 2008-02-20 | 2009-08-26 | 北京师范大学 | Method for extracting and purifying zymoprotein anaerobically degraded by polycyclic aromatic hydrocarbon |
| CN101974443A (en) * | 2010-03-02 | 2011-02-16 | 北京师范大学 | Benzo[a]pyrene-degrading strain and application thereof |
| CN102533578A (en) * | 2010-12-10 | 2012-07-04 | 中国农业大学 | Microorganism bacterium agent for degrading polycyclic aromatic hydrocarbon |
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