CN1746293A - Engineering bacteria for degrading pyrene gene efficiently and construction thereof - Google Patents

Abstract

A high-efficient degradable gene engineering bacterium and its construction are disclosed. The engineering bacterium is prepared by using cytochrome P450 mono-oxygenase pc-1 gene of Phanerochaete chrysosporium, reverse-DNA augmentation reacting to obtain pc-1 cDNA, converting cDNA into colibacillus JM109 and constructing to high-efficient functional gene engineering bacterium Jm413. It has better adaptation and safety, it can be used for biological repair and restoration.

Description

Engineering bacteria for degrading pyrene gene efficiently and structure thereof

Technical field

The present invention relates to biological restoration or ecological recovery field, specifically a kind of engineering bacteria for degrading pyrene gene and structure thereof.

Background technology

Rate-limiting step is the phenyl ring oxygenation of beginning in degrading polycyclic aromatic hydrocarbons, in case finish the phenyl ring oxygenation, reoxidizes degraded subsequently and just relatively easily carries out.But common microorganism often lacks monooxygenase gene, and perhaps monooxygenase can not be expressed.

Summary of the invention

The purpose of this invention is to provide a kind of engineering bacteria for degrading pyrene gene efficiently and construction process thereof.

Engineering bacteria for degrading pyrene gene efficiently of the present invention is the cytochrome P 450 monooxygenases pc-1 gene with white-rot fungi (Phanerochaete chrysosporium), obtain the cDNA (complementary DNA) of pc-1 through reverse transcription-dna amplification reaction (RT-PCR), again cDNA is transformed in the e. coli jm109, be built into the genetic engineering bacterium of function efficient degradation polycyclic aromatic hydrocarbons, called after JM413.The inventor guaranteed to provide this project bacterium to the public in 20 years applyings date.White-rot fungi (Phanerochaetechrysosporium) is purchased in Microbe Inst., Chinese Academy of Sciences's bacterial classification and is preserved the center.

The construction process of engineering bacteria for degrading pyrene gene efficiently,

1) design of primers is according to the gene order of the cytochrome P 450 monooxygenases pc-1 of white-rot fungi (Phanerochaete chrysosporium), choose its upstream and downstream conserved sequence design upstream and downstream primer A and B, and add the restriction enzyme site sequence of EcoR I and two restriction enzymes of BamH I at an end of each primer respectively.The nucleotide sequence of primer A and B is as follows, and underscore partly is the restriction enzyme site sequence of EcoR I and two restriction enzymes of BamH I.

Primer A:5 '-ATG GAA TTCATG GTG ACT ACT TTT ACG AG-3 '

Primer B:5 '-ATG GGA TCCGGA TTG CTT CTG C-3 '

2) extract the cell extraction total RNA of total RNA from white rot fungi (Phanerochaete chrysosporium)

3) reverse transcription-dna amplification reaction (RT-PCR) adopts the method for an one step RT-PCR, begins reverse transcription-the amplify cDNA of monooxygenase gene pc-1 by total RNA

4) cDNA of carrier pUC18 and monooxygenase pc-1 is hybridized under the effect of ligase enzyme is a new plasmid to recombinant plasmid.

5) transform will hybridization novel plasmid transform in the e. coli jm109.(e. coli jm109 is purchased in Huamei Bio-Engrg Co.)

6) called after JM413 has been advanced in the e. coli jm109 in the cDNA gene transformation of screening and differentiate further screening of warp and discriminating confirmation form oxygenase pc-1.

7) performance measurement experimental results show that with the oxidase activity of pyrogallol spectrophotometry genetic engineering bacterium JM413 the JM413 oxidase activity improves 135% than JM109; With typical polycyclic aromatic hydrocarbon compounds-pyrene is the degraded target compound, the degradation characteristic of check genetic engineering bacterium JM413, and the result shows, under identical condition, the pyrene degradation rate of JM413 is nearly 20 times of JM109 behind bacterium and pyrene effect 48h.

The present invention is with the gene of the cytochrome P 450 monooxygenases pc-1 of eukaryote white-rot fungi (Phanerochaete chrysosporium), obtain cDNA through the RT-PCR technology, again cDNA is transformed in the prokaryotic organism e. coli jm109, successfully construct genetic engineering bacterium JM413 with monooxygenase activity.The genetic engineering bacterium JM413 that makes up except that the characteristic with JM109, also has monooxygenase gene, can efficiently express to produce oxidasic characteristic.This bacterium has the characteristic of efficient degradation polycyclic aromatic hydrocarbons, has significant application value and vast market prospect in biological restoration or ecological recovery engineering.

Beneficial effect of the present invention:

The genetic engineering bacterium JM413 that makes up, because of having ampicillin resistance gene and monooxygenase gene, so adaptability is strong, easy enlarged culturing, and safe, can not cause any detrimentally affect, therefore to environment, can be widely used in biological restoration and ecological recovery engineering, the degraded hardly degraded organic substance promotes ecological recovery, keeps ecological balance.

Test, assay:

(1). the oxidase activity of genetic engineering bacterium is measured

1. experimental principle

The pyrogallol colorimetry based on being matrix with pyrogallol (claiming pyrogallol again), is having under the situation of atmospheric oxygen, because the oxidasic catalysis of microorganism generates colored Nutgalls element.Produce the Nutgalls element and maximum light absorption value is arranged, its content and color depth (A under the 610nm wavelength _610nm) be proportionate.With 7.5mg.mL ^-1The color of potassium dichromate aqueous solution is corresponding to 1mg.mL ^-1The ethyl acetate solution of Nutgalls element.Show oxidase activity with the scale that generates the Nutgalls element in the unit time.

2. measurement result (table 1-1), histogram (Fig. 1-1)

Table 1-1 JM413 and JM109 oxidase activity

The measurement result explanation, the oxidase activity of genetic engineering bacterium JM413 is a JM109 oxidase activity 135%.This is owing to white-rot fungi cytochrome P 450 monooxygenases pc-1 gene has been advanced in conversion in the reorganization bacterium, and has obtained efficiently expressing, and has improved the result of the oxidation capacity of JM413.

(2). genetic engineering bacterium JM413 and JM109 are to the comparison of pyrene degradation characteristic

1, the preparation of bacteria suspension:

Each picking one ring thalline joins respectively the 10ml LB nutrient solution from the preservation inclined-plane of JM109 and JM413, at 37 ℃ of following 200rmin ^-1Shaking culture is spent the night, and respectively its whole switchings is gone in the fresh LB nutrient solution of 90ml 37 ℃ of following 200rmin ^-1The about 24h of constant-temperature shaking culture is to logarithmic phase A ₆₀₀(the every mL bacterium liquid colony number about 8.0 * 10 that is about at 2.0 o'clock ⁸), the centrifugal 10min of 4000rpm, 4 ℃, refrigerator is preserved standby.

2. degradation experiment

Pipette pyrene-acetone soln (1gL of 200 μ L ^-1) to the 100ml Erlenmeyer flask,, under aseptic condition, adding JM109 and JM413 bacteria suspension 4mL respectively to wherein adding 36mL sterilization inorganic salt nutrient solution, final pyrene concentration is 5mgL ^-1, 37 ℃ of 200rmin ^-1Shaking culture.Timing sampling is measured the concentration of pyrene in the solution.

3. pyrene Determination on content

(1) get the bacterium degradation solution of cultivating certain hour, on Bechtop, it is all transferred in the 50mL centrifuge tube, the centrifugal 10min of 4800rpm, supernatant liquor adds the 20mL ethyl acetate, and precipitation adds the 10mL ethyl acetate, and 0.5h is extracted in the room temperature vibration.

(2) ethyl acetate and supernatant liquor all are transferred in the separating funnel, the violent mixing, leave standstill 10min, after treating layering, collect ethyl acetate layer (upper strata), add the 10mL ethyl acetate again to aqueous phase, repeat said extracted step 3 time, collect ethyl acetate layer, be settled to 100mL.

(3) will precipitate and use ethyl acetate extraction, the centrifugal 10min of 4800rpm.Ethyl acetate is transferred in the 50mL volumetric flask, extracts repeatedly three times.Last united extraction liquid ethyl acetate is settled to 50mL.

(4) acetic acid ethyl acetate extract is used the rotatory evaporator evaporate to dryness, be settled to 10mL with hplc grade methanol, with the special-purpose membrane filtration (0.45 μ m) of liquid chromatography, high pressure lipuid chromatography (HPLC) (HPLC) is measured the concentration of pyrene.

(5) high pressure lipuid chromatography (HPLC): Waters1525 type high pressure liquid chromatograph, Waters 2475Multi λ Fluorescence Detector (fluorimetric detector), Waters Symmetry Shield ^TMRP ₁₈Chromatographic column (5 μ m, 3.9 * 150mm), moving phase is acetonitrile/H ₂(v: v=80: 20), flow velocity is 1mLmin to O ^-1The fluorimetric detector excitation wavelength is 333nm, and the fluorescent emission wavelength is 390nm, and sensitivity is 2.0, and sample size is 20 μ l, measures under the room temperature, and external standard method is quantitative.Detect and be limited to 0.5 μ gL ^-1

4. measurement result (table 1-2, Fig. 1-2, Fig. 1-3)

Table 1-2 pyrene degraded test-results

Degradation time (h)		12	24	36	48
						JM109 thalline adsorptive capacity (μ g)	Sample 1	51.28	43.78	81.90	81.89
Sample 2	59.88	58.88	80.07	79.21
					On average		55.58	51.33	80.83	80.56
Pyrene content in the JM109 nutrient solution (μ g)		123.48	128.77	98.01							99.72

JM413 thalline adsorptive capacity (μ g)	Sample 1	111.27	118.02	118.09	110.21
						Sample 2	105.40	106.02	125.08	115.40
	On average	108.34	112.02	121.58	112.79
						Pyrene content in the JM413 nutrient solution (μ g)		48.07	38.72	25.74	16.70
The add-on of pyrene (μ g)		200	200	200	200
						The yield of pyrene (μ g)		183.34	183.34	183.34	183.34
The adsorption rate of JM109 pyrene (%)		30.31	27.99	44.08	43.94
						The adsorption rate of JM413 pyrene (%)		59.08	61.09	66.30	61.52
The degradation rate of JM109 pyrene (%)		14.70	17.80	19.65	29.37
						The degradation rate of JM413 pyrene (%)		2.35	1.78	2.47	1.67

Description of drawings

Fig. 1-the 1st, the oxidase activity comparison diagram

Fig. 1-2 is the graphic representation of JM413 degraded pyrene

Fig. 1-the 3rd, JM109 and JM413 degradation amount comparison diagram

Embodiment

Embodiment 1

(1) extraction of total RNA

1) white-rot fungi is cultured to logarithmic phase with potato culture, gets thalline 50mg and add 500 μ L denaturing solns and 3.6 μ L beta-mercaptoethanols, mixing is bathed homogenate with the homogenate ice-cream stick.

2). add immediately 100 μ L sodium-acetates (2mol/L, pH4.0), 200 μ L water-saturated phenols and 200 μ L chloroform/primary isoamyl alcohol (24: 1).After adding every kind of component, cover centrifuge tube lid, the abundant mixing of jolting gently, ice bath 15min.

3). at 4 ℃ of centrifugal 10min of following 10000rpm, the water that will contain RNA moves in the new centrifuge tube.

4). add and the isopyknic Virahol of extracting solution, thorough mixing is in-20 ℃ of precipitated rna 1h or longer time.At 4 ℃ of centrifugal 15min of following 15000rpm

5). decant Virahol, add 100 μ l dissolving RNA particle, and add the equal-volume Virahol, in-20 ℃ of precipitated rna 1h or longer time.

6). in 4 ℃ of centrifugal 15min of following 15000rpm, collect the RNA precipitation.Heavy centrifugal 2 times of ethanol repeated washing with 75%.The ethanol that sucking-off is remaining, and uncap inversion several minutes are controlled dried ethanol.

7). add 100 μ LDEPC treated water dissolution precipitations, total rna solution is stored in-70 ℃.(all centrifuge tube samplers all will be handled with DEPC in the experiment).

(2) reverse transcription-DNA cloning (RT-PCR)

Contaminated for preventing RNA, reaction is all operated under aseptic condition

1). get the special-purpose 0.5mL thin-walled centrifuge tubes of 5 PCR (2 samples, 3 contrasts) and place on ice, add

2*RT-PCR buffered soln 25 μ L

Template ribonucleic acid (total RNA) final concentration 10pg-1ug

Primer A 1 μ L

Primer B 1 μ L

RT/Taq Mix 1μL

Aseptic double-distilled water complements to 50 μ L

Negative control:

Contrast 1: do not add template ribonucleic acid

Contrast 2: do not add ThermoScript II and substitute RT/Taq Mix with 2U Taq

Contrast 3: do not add primer

2). mixing gently, (moment is centrifugal, guarantees that all components is at the pipe end), on cover the about 50 μ l of whiteruss.

3) .RT-PCR step and condition

The 1st circulation	37 ℃ of synthetic 15-30min of cDNA, 94 ℃ of pre-sex change 2min
		35-40 cycle P CR amplification	94 ℃ of sex change 15s, 55-60 ℃ of annealing 30s, 68-72 ℃ is extended 40s
Last 1 circulation	72 ℃ are extended 5-10min, obtain cDNA

(3) carrier puc18DNA cuts with the enzyme of cDNA and is connected

1). enzyme is cut

(1) the cDNA enzyme is cut:

BamH I 0.8μL

EcoR I 0.8μL

Buffer K 2.0μL

(final concentration is 0.1 μ g μ L to the about 1.0 μ L of cDNA ^-1)

Aseptic double-distilled water H ₂O complements to 20 μ L

37 ℃ of water bath with thermostatic control 1h add 50 μ L dehydrated alcohols, place more than the 30min for-20 ℃, and 4 ℃, the centrifugal 10min of 15000rpm, abandoning supernatant is inverted 20min, adds aseptic double-distilled water 10 μ L.

(2) enzyme of carrier puc18DNA is cut:

BamH I 0.8μL

EcoR I 0.8μL

Buffer K 2.0μL

(final concentration is 0.1 μ g μ L to the about 1.0 μ L of pUC18DNA ^-1)

DdH ₂O complements to 20 μ L

37 ℃ of water bath with thermostatic control 1h add 50 μ l dehydrated alcohols, place more than the 30min for-20 ℃, and 4 ℃, the centrifugal 10min of 15000rpm, abandoning supernatant is inverted 20min, adds aseptic double-distilled water 10 μ L ,-20 ℃ of preservations.

2) cDNA and pUC18DNA's is connected

Target DNA 3.0 μ L

Carrier DNA 1.0 μ L

T ₄Ligase enzyme 1.0 μ L

10*Buffer 1.0μL

Aseptic double-distilled water complements to 10.0 μ L

16 ℃ of constant temperature spend the night ,-20 ℃ of preservations.

(4) preparation of competent cell and conversion

1) get 100 μ L bacterial strain E.coli JM109 glycerine and preserve bacterium liquid, insert in the 3mL LB liquid medium, 37 ℃ of joltings are spent the night, and nutrient solution is changed in the fresh LB nutrient solution of 25mL, and 37 ℃ are continued to be cultured to A ₆₀₀Be about 0.3～0.4, get the 1.5mL nutrient solution in 4 ℃ of centrifugal 10min of following 5000rpm, supernatant discarded is inverted control and is done.

2) add 0.5mL 0.1molL ^-1CaCl ₂-MgCl ₂, ice bath 10min is in 4 ℃ of centrifugal 10min of following 10000rpm, supernatant discarded.

3). in thalline, add 1ml 0.1molL again ^-1CaCl ₂-MgCl ₂, mix, be distributed into 100 μ L/ pipe ,-20 ℃ of preservations.

4). get cDNA and join in the 100 μ L competent cells with the liquid 5 μ L that are connected of pUC18DNA, behind the ice bath 30min, 42 ℃ of heat shock 90s, ice bath 1～2min immediately after the taking-up.

5). contain the LB nutrient solution of penbritin (0.1%), 37 ℃ of 70rmin to wherein adding 800 μ L ^-1Constant temperature culture 45min, 4 ℃, the centrifugal 10min of 10000rpm discards the part supernatant liquor, and-20 ℃ of preservations are standby.

(5) with X-gal and IPTG screening positive recombinant bacterium colony---blue hickie screening

1). drip 40 μ L 2%X-gal and 8 μ L 20%IPTG in the dull and stereotyped central authorities of the prefabricated LB that contains penbritin.

2). the spreader with a sterilization is even with X-gal and IPTG solution coat, makes it to be dispersed in media surface.Place 0.5h to the media surface absence of liquid for 37 ℃.

3). inoculate the transformed bacteria suspension of 100 μ L (four) preparation, with aseptic spreader coating evenly, treat that inoculation liquid absorbs fully after, the inversion culture plate is in 37 ℃ of overnight incubation.

4). take out culture plate in 4 ℃ of placement 0.5～1h, make the bacterium colony colour developing fully.

5). filter out the bacterium colony of white, be genetic engineering bacterium, called after JM413.

Embodiment two

(1) extraction of total RNA

1) white-rot fungi is cultured to logarithmic phase with potato culture, gets thalline 100mg500 μ L denaturing soln and 3.6 μ L beta-mercaptoethanols, mixing is bathed homogenate with the homogenate ice-cream stick.

2). add immediately 100 μ L sodium-acetates (2mol/L, pH4.0), 200 μ L water-saturated phenols and 200 μ L chloroform/primary isoamyl alcohol (24: 1).After adding every kind of component, cover centrifuge tube lid, the abundant mixing of jolting gently, ice bath 15min.

3). at 4 ℃ of centrifugal 10min of following 10000rpm, the water that will contain RNA moves in the new centrifuge tube.

4). add and the isopyknic Virahol of extracting solution, thorough mixing is in-20 ℃ of precipitated rna 1h or longer time.At 4 ℃ of centrifugal 15min of following 15000rpm

5). decant Virahol, add 100 μ l dissolving RNA particle, and add the equal-volume Virahol, in-20 ℃ of precipitated rna 1h or longer time.

6). in 4 ℃ of centrifugal 15min of following 15000rpm, collect the RNA precipitation.Heavy centrifugal 2 times of ethanol repeated washing with 75%.The ethanol that sucking-off is remaining, and uncap inversion several minutes are controlled dried ethanol.

7). add 100 μ LDEPC treated water dissolution precipitations, total rna solution is stored in-70 ℃.(all centrifuge tube samplers all will be handled with DEPC in the experiment).

(2) reverse transcription-DNA cloning (RT-PCR)

Contaminated for preventing RNA, reaction is all operated under aseptic condition

1). get the special-purpose 0.5mL thin-walled centrifuge tubes of 5 PCR (2 samples, 3 contrasts) and place on ice, add

2*RT-PCR buffered soln 25 μ L

Template ribonucleic acid (total RNA) final concentration 10pg～1ug

Primer A 2 μ L

Primer B 2 μ L

RT/Taq Mix 2μL

Aseptic double-distilled water complements to 50 μ L

Negative control:

Contrast 1: do not add template ribonucleic acid

Contrast 2: do not add ThermoScript II and substitute RT/Taq Mix with 2U Taq

Contrast 3: do not add primer

2). mixing gently, (moment is centrifugal, guarantees that all components is at the pipe end), on cover the about 50 μ l of whiteruss.

3) .RT-PCR step and condition

The 1st circulation	37 ℃ of synthetic 95 ℃ of pre-sex change 2min of 15～30min of cDNA
		The 35th～40 cycle P CR amplification	95 ℃ of sex change 15s, 55 ℃ of annealing 30s, 72 ℃ are extended 40s
Last 1 circulation	72 ℃ are extended 5-10min, obtain cDNA

(3) carrier puc18DNA cuts with the enzyme of cDNA and is connected

1). enzyme is cut

(1) the cDNA enzyme is cut:

BamH I 0.8μL

EcoR I 0.8μL

Buffer K 2.0μL

(final concentration is 0.1 μ g μ L to the about 1.0 μ L of cDNA ^-1)

Aseptic double-distilled water H ₂O complements to 20 μ L

37 ℃ of water bath with thermostatic control 1h add 50 μ L dehydrated alcohols, place more than the 30min for-20 ℃, and 4 ℃, the centrifugal 10min of 15000rpm, abandoning supernatant is inverted 20min, adds aseptic double-distilled water 10 μ L.

(2) enzyme of carrier puc18DNA is cut:

BamH I 0.8μL

EcoR I 0.8μL

Buffer K 2.0μL

(final concentration is 0.1 μ g μ L to the about 1.0 μ L of pUC18 DNA ^-1)

DdH ₂O complements to 20 μ L

37 ℃ of water bath with thermostatic control 1h add 50 μ l dehydrated alcohols, place more than the 30min for-20 ℃, and 4 ℃, the centrifugal 10min of 15000rpm, abandoning supernatant is inverted 20min, adds aseptic double-distilled water 10 μ L ,-20 ℃ of preservations.

2) cDNA and pUC18DNA's is connected

Target DNA 3.0 μ L

Carrier DNA 1.0 μ L

T ₄Ligase enzyme 1.0 μ L

10*Buffer 1.0μL

Aseptic double-distilled water complements to 10.0 μ L

16 ℃ of constant temperature spend the night ,-20 ℃ of preservations.

(4) preparation of competent cell and conversion

1) get 100 μ L bacterial strain E.coli JM109 glycerine and preserve bacterium liquid, insert in the 3mL LB liquid medium, 37 ℃ of joltings are spent the night, and nutrient solution is changed in the fresh LB nutrient solution of 25mL, and 37 ℃ are continued to be cultured to A ₆₀₀Be about 0.3～0.4, get the 1.5mL nutrient solution in 4 ℃ of centrifugal 10min of following 5000rpm, supernatant discarded is inverted control and is done.

2) add 0.5mL 0.1molL ^-1CaCl ₂-MgCl ₂, ice bath 10min is in 4 ℃ of centrifugal 10min of following 10000rpm, supernatant discarded.

3). in thalline, add 1ml 0.1molL again ^-1CaCl ₂-MgCl ₂, mix, be distributed into 100 μ L/ pipe ,-20 ℃ of preservations.

4) cDNA joins in the 100 μ L competent cells with the liquid 5 μ L that are connected of pUC18DNA, behind the ice bath 30min, and 42 ℃ of heat shock 90s, ice bath 1～2min immediately after the taking-up.

5) wherein add the LB nutrient solution that 800 μ L contain penbritin (0.1%), 37 ℃ of 70rmin ^-1Constant temperature culture 45min, 4 ℃, the centrifugal 10min of 10000rpm discards the part supernatant liquor, and-20 ℃ of preservations are standby.

(5) with X-gal and IPTG screening positive recombinant bacterium colony---blue hickie screening

1). drip 40 μ L 2%X-gal and 8 μ L 20%IPTG in the dull and stereotyped central authorities of the prefabricated LB that contains penbritin.

2). the spreader with a sterilization is even with X-gal and IPTG solution coat, makes it to be dispersed in media surface.Place 0.5h to the media surface absence of liquid for 37 ℃.

3). inoculate the transformed bacteria suspension of 100 μ L (four) preparation, with aseptic spreader coating evenly, treat that inoculation liquid absorbs fully after, the inversion culture plate is in 37 ℃ of overnight incubation.

4) culture plate is placed 0.5～1h in 4 ℃, make the bacterium colony colour developing fully.

5). filter out the bacterium colony of white, be genetic engineering bacterium, called after JM413.

Embodiment three

(1) extraction of total RNA

1) white-rot fungi is cultured to logarithmic phase with potato culture, gets thalline 80mg500 μ L denaturing soln and 3.6 μ L beta-mercaptoethanols, mixing is bathed homogenate with the homogenate ice-cream stick.

2). add immediately 100 μ L sodium-acetates (2mol/L, pH4.0), 200 μ L water-saturated phenols and 200 μ L chloroform/primary isoamyl alcohol (24: 1).After adding every kind of component, cover centrifuge tube lid, the abundant mixing of jolting gently, ice bath 15min.

3). at 4 ℃ of centrifugal 10min of following 10000rpm, the water that will contain RNA moves in the new centrifuge tube.

4). add and the isopyknic Virahol of extracting solution, thorough mixing is in-20 ℃ of precipitated rna 1h or longer time.At 4 ℃ of centrifugal 15min of following 15000rpm

5). decant Virahol, add 100 μ l dissolving RNA particle, and add the equal-volume Virahol, in-20 ℃ of precipitated rna 1h or longer time.

6). in 4 ℃ of centrifugal 15min of following 15000rpm, collect the RNA precipitation.Heavy centrifugal 2 times of ethanol repeated washing with 75%.The ethanol that sucking-off is remaining, and uncap inversion several minutes are controlled dried ethanol.

7). add 100 μ LDEPC treated water dissolution precipitations, total rna solution is stored in-70 ℃.(all centrifuge tube samplers all will be handled with DEPC in the experiment).

(2) reverse transcription-DNA cloning (RT-PCR)

Contaminated for preventing RNA, reaction is all operated under aseptic condition

1). get the special-purpose 0.5mL thin-walled centrifuge tubes of 5 PCR (2 samples, 3 contrasts) and place on ice, add

2*RT-PCR buffered soln 25 μ L

Template ribonucleic acid (total RNA) final concentration 10pg～1ug

Primer A 2 μ L

Primer B 2 μ L

RT/Taq Mix 2μL

Aseptic double-distilled water complements to 50 μ L

Negative control:

Contrast 1: do not add template ribonucleic acid

Contrast 2: do not add ThermoScript II and substitute RT/Taq Mix with 2U Taq

Contrast 3: do not add primer

2). mixing gently, (moment is centrifugal, guarantees that all components is at the pipe end), on cover the about 50 μ l of whiteruss.

3) .RT-PCR step and condition

The 1st circulation	37 ℃ of synthetic 95 ℃ of pre-sex change 3min of 15～30min of cDNA
		The 35th～40 cycle P CR amplification	95 ℃ of sex change 15s, 55 ℃ of annealing 30s, 72 ℃ are extended 40s
Last 1 circulation	72 ℃ are extended 5～8min and obtain cDNA

(3) carrier puc18DNA cuts with the enzyme of cDNA and is connected

1). enzyme is cut

(1) the cDNA enzyme is cut:

BamH I 0.8μL

EcoR I 0.8μL

Buffer K 2.0μL

(final concentration is 0.1 μ g μ L to the about 1.0 μ L of cDNA ^-1)

Aseptic double-distilled water H ₂O complements to 20 μ L

37 ℃ of water bath with thermostatic control 1h add 50 μ L dehydrated alcohols, place more than the 30min for-20 ℃, and 4 ℃, the centrifugal 10min of 15000rpm, abandoning supernatant is inverted 20min, adds aseptic double-distilled water 10 μ L.

(2) enzyme of carrier puc18DNA is cut:

BamH I 0.8μL

EcoR I 0.8μL

Buffer K 2.0μL

(final concentration is 0.1 μ g μ L to the about 1.0 μ l of pUC18 DNA ^-1)

DdH ₂O complements to 20 μ L

37 ℃ of water bath with thermostatic control 1h add 50 μ l dehydrated alcohols, place more than the 30min for-20 ℃, and 4 ℃, the centrifugal 10min of 15000rpm, abandoning supernatant is inverted 20min, adds aseptic double-distilled water 10 μ L ,-20 ℃ of preservations.

2) cDNA and pUC18DNA's is connected

Target DNA 3.0 μ L

Carrier DNA 1.0 μ L

T ₄Ligase enzyme 1.0 μ L

10*Buffer 1.0μL

Aseptic double-distilled water complements to 10.0 μ L

16 ℃ of constant temperature spend the night ,-20 ℃ of preservations.

(4) preparation of competent cell and conversion

1) get 100 μ L bacterial strain E.coli JM109 glycerine and preserve bacterium liquid, insert in the 3mL LB liquid medium, 37 ℃ of joltings are spent the night, and nutrient solution is changed in the fresh LB nutrient solution of 25mL, and 37 ℃ are continued to be cultured to A ₆₀₀Be about 0.3～0.4, get the 1.5mL nutrient solution in 4 ℃ of centrifugal 10min of following 5000rpm, supernatant discarded is inverted control and is done.

2) add 0.5mL 0.1molL ^-1CaCl ₂-MgCl ₂, ice bath 10min is in 4 ℃ of centrifugal 10min of following 10000rpm, supernatant discarded.

3). in thalline, add 1ml 0.1molL again ^-1CaCl ₂-MgCl ₂, mix, be distributed into 100 μ L/ pipe ,-20 ℃ of preservations.

4) cDNA joins in the 100 μ L competent cells with the liquid 5 μ L that are connected of pUC18DNA, behind the ice bath 30min, and 42 ℃ of heat shock 90s, ice bath 1～2min immediately after the taking-up.

5) contain the LB nutrient solution of penbritin (0.1%), 37 ℃ of 70rmin to wherein adding 800 μ L ^-1Constant temperature culture 45min, 4 ℃, the centrifugal 10min of 10000rpm discards the part supernatant liquor, and-20 ℃ of preservations are standby.

(5) with X-gal and IPTG screening positive recombinant bacterium colony---blue hickie screening

1). drip 40 μ L 2%X-gal and 8 μ L 20%IPTG in the dull and stereotyped central authorities of the prefabricated LB that contains penbritin.

2). the spreader with a sterilization is even with X-gal and IPTG solution coat, makes it to be dispersed in media surface.Place 0.5h to the media surface absence of liquid for 37 ℃.

3). inoculate the transformed bacteria suspension of 100 μ L (four) preparation, with aseptic spreader coating evenly, treat that inoculation liquid absorbs fully after, the inversion culture plate is in 37 ℃ of overnight incubation.

4) culture plate is placed 0.5～1h in 4 ℃, make the bacterium colony colour developing fully.

5) filter out white bacterium colony, be genetic engineering bacterium, called after JM413.

Sequence list

SEQUENCE LISTING

＜110〉Nankai University

＜120〉engineering bacteria for degrading pyrene gene efficiently and structure thereof

<130>20050720

<160>2

<170>PatentIn version 3.1

<210>1

<211>29

<212>DNA

<213>JM413

<220>

<221>primer_bind

<222>(1)..(29)

<223>

<400>1

atggaattca tggtgactac ttttacgag 29

<210>2

<211>22

<212>DNA

<213>JM413

<220>

<221>primer_bind

<222>(1)..(22)

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atgggatccg gattgcttct gc 22

Claims (2)

1. engineering bacteria for degrading pyrene gene efficiently, it is characterized in that, it is the cytochrome P 450 monooxygenases pc-1 gene with white-rot fungi (Phanerochaetechrysosporium), obtain the cDNA of pc-1 through reverse transcription-dna amplification reaction, again cDNA is transformed in the e. coli jm109, be built into the genetic engineering bacterium of function efficient degradation polycyclic aromatic hydrocarbons, called after JM413.

2. the construction process of an engineering bacteria for degrading pyrene gene efficiently is characterized in that, comprises the steps:

1) design of primers is according to the gene order of the cytochrome P 450 monooxygenases pc-1 of white-rot fungi (Phanerochaete chrysosporium), choose its upstream and downstream conserved sequence design upstream and downstream primer A and B, and add the restriction enzyme site sequence of EcoRI and two restriction enzymes of BamHI at an end of each primer respectively.The nucleotide sequence of primer A and B is as follows, and underscore partly is the restriction enzyme site sequence of EcoRI and two restriction enzymes of BamHI;

Primer A:5 '-ATG GAA TTCATG GTG ACT ACT TTT ACG AG-3 '

Primer B:5 '-ATG GGA TCCGGA TTG CTT CTG C-3 '

2) extract the cell extraction total RNA of total RNA from white rot fungi (Phanerochaete chrysosporium);

3) reverse transcription-dna amplification reaction (RT-PCR) adopts the method for an one step RT-PCR, begins reverse transcription-the amplify cDNA of monooxygenase gene pc-1 by total RNA;

4) cDNA of carrier pUC18 and monooxygenase pc-1 is hybridized under the effect of ligase enzyme is a new plasmid to recombinant plasmid;

5) transform will hybridization novel plasmid transform in the e. coli jm109;

6) called after JM413 has been advanced in the e. coli jm109 in the cDNA gene transformation of screening and differentiate further screening of warp and discriminating confirmation form oxygenase pc-1.

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