CN102373202A - Biphenyl degradation related gene operon derived from fluorescent pseudomonas and expression vector and application thereof - Google Patents

Abstract

The invention relates to a gene operon related to biphenyl degradation derived from Pseudomonas fluorescens, its expression vector and application. The base sequence of the biphenyl degradation-related gene operon derived from Pseudomonas fluorescens is shown in SEQ ID No 1, which contains 1313 bases A, 1955 bases C, and 2117 bases G, Base T 1331. The biphenyl degradation-related gene operon derived from Pseudomonas fluorescens and its expression vector can be used for biodegradation of biphenyl and polychlorinated biphenyl.

Description

Biphenyl degradation-related gene operon derived from Pseudomonas fluorescens and its expression vector and application

technical field

The invention belongs to the field of microbial genetic engineering, in particular to a biphenyl degradation-related gene operon derived from Pseudomonas fluorescens and its expression vector, in particular to a biphenyl degradation-related gene derived from Pseudomonas fluorescens Application of operon and its expression vector in degrading biphenyl and polychlorinated biphenyl.

Background technique

Polychlorinated biphenyls (PCBs), also known as chlorinated biphenyls, are a class of artificially synthesized organic compounds, a class of chlorides formed by replacing hydrogen atoms on the biphenyl ring with chlorine atoms. Since the first successful synthesis of PCBs in 1881, 209 such compounds have been artificially synthesized. PCBs are semi-volatile or non-volatile substances with strong corrosiveness. PCBs have the characteristics of typical persistent organic pollutants, that is, refractory and long-distance migration, which pose a greater threat to human health [Furukawa and Gen, Appl Microbiol, 2000, 46: 283-296; Wiegel and Wu, FEMS Microbiology Ecology, 2000, 32, 1-15; Jim and Reyes, Environmental Pollution, 2008, 155: 1-12].

Currently, many strains that degrade PCBs have been isolated. Ahmed and Focht isolated for the first time two colorless strains (Achromobacter), which can degrade some PCBs compounds into chlorobenzoic acid [Ahmed and Focht, Can JMicrobiol, 1973, 19:47-52]. Then Furukawa and Matsumura reported the degradation of 31 different PCBs compounds using 2 biphenyl-degrading bacteria [Furukawa and Matsumura, J Agric Food Chem, 1976, 24:251-256]. Pseudomonas pseudoalcaligenes KF707 was isolated from the soil near a biphenyl production plant in Kitakyushu, Japan. This strain has a narrow range of degradation of PCBs [Furukawa et al., JBacteriol, 1986, 166: 392-398]. Burkholderia cepacia LB400 was isolated from a PCB-contaminated site in New York, USA [Bedard et al., Appl. . The experiment on PCBs degraded by LB400 strain showed that LB400 degraded PCBs in a larger range than KF707. Tests of 19 different PCBs isomers showed that LB400 can degrade 17 species, while KF707 only degrades 8 species [Erickson and Mondello, Appl Environ Microbiol, 1993, 59:3858-62; Gibson et al., J Bacteriol, 1993 , 175: 4561-4564]. Jia et al. isolated a Gram-negative bacterium with a relatively strong ability to degrade PCBs, named LY402. LY402 can degrade high-chlorinated biphenyl strains, which is different from the strains isolated in the past [Jia et al., J Microbiol Biotechnol, 2008, 18: 952-957]. In addition, some fungi, yeast, and cyanobacteria can degrade low-chloride PCBs into mono- and di-hydroxy compounds. For example, a white-rot fungus Phanerochaete chrysosporium has a high degradation efficiency for low-chlorine PCBs [Dietrich et al., Appl Environ Microbiol, 1995, 61: 3904-3909; Beaudette et al., Appl Environ Microbiol, 1998, 64, 2020-2025] . The degradation of PCBs is mainly composed of biphenyl dioxygenase (BphA), dihydrodihydroxy dehydrogenase (BphB), 2,3 dihydroxy dioxygenase encoded by the four genes bphA, bphB, bphC and bphD of the bph gene cluster The enzyme (BphC) and the hydrolase (BphD) work together [Furukawa, Biodegradation, 1994, 5: 289-300; Masayuki et al., J Biosci Bioeng, 2002, 421-427].

Contents of the invention

One of the technical problems to be solved by the present invention is to provide a biphenyl degradation-related gene operon of Pseudomonas fluorescens, which is derived from Pseudomonas sp.

The second technical problem to be solved by the present invention is to provide an expression vector comprising a Pseudomonas fluorescens biphenyl degradation-related gene operon.

The third technical problem to be solved by the present invention is to provide an application of an expression vector comprising a Pseudomonas fluorescens biphenyl degradation-related gene operon in the biodegradation of biphenyl and polychlorinated biphenyl.

The present invention achieves the above object through the following technical solutions.

The biphenyl degradation-related gene operon is derived from Pseudomonas fluorescens, and its base sequence is shown in SEQID No 1, which contains 1313 bases A, 1955 bases C, 2117 bases G, and 2117 bases G. Base T 1331.

The preparation method of the biphenyl degradation-related gene operon derived from Pseudomonas fluorescens comprises the following steps:

1) Screening of strains

The soil samples were selected from a 10-meter river silt near an abandoned chemical plant in Laogang, Pudong New Area (formerly Nanhui District), Shanghai. Put the soil sample into a sterilized paper bag, weigh 10g of the soil sample on an ultra-clean workbench, put it into a ceramic mortar, add 50mL of sterile water, mash it with a sterile pestle, and let it stand for 5 minutes. Aspirate 1-5mL supernatant and add to 100mL M9 liquid basic medium containing 0.01wt% trichlorobiphenyl as the sole carbon source, culture at 28°C at 150 rpm for 3 days, take 1mL of it and add it to a new 100mL containing 0.01 Continue to subculture in the M9 liquid basic medium with wt% trichlorobiphenyl as the only carbon source. On the culture medium, cultured at 28°C for 3 days, and a well-growing Pseudomonas fluorescens was selected for further research.

2) Extraction of the total DNA of the strain

The isolated Pseudomonas fluorescens strain was cultured overnight (16 hours) at 28°C and 150 rpm in 100 mL of liquid LB medium, and the cell culture solution was centrifuged at 6,000 g for 5 min to obtain cell pellets. These pellets were frozen at -20°C for 1 h. Then use TE (10mM Tris-HCl, 1mM EDTA, pH 8.0) solution to wash once. Add sterile water with a concentration of 10 mg/mL lysozyme to suspend, and incubate on a shaking table at 37° C. for 1 h. Add 0.5M EDTA, 10% (w/v) SDS and 5M NaCl and mix gently by shaking. Then protein K was added at a concentration of 20 mg/mL, and the reaction was incubated at 37° C. for 1 h. DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) equivalent to the volume of the culture liquid (1 volume). The aqueous phase was extracted with chloroform:isoamyl alcohol (24:1) corresponding to 1/2 (0.5 volume) of the volume of the aqueous phase. Shake to mix and centrifuge for 5 min. Add isoamyl alcohol equivalent to the volume of the aqueous phase (1 volume) into the aqueous phase. Shake to mix and centrifuge for 15 min. The pellet was taken, the DNA was rinsed with 70% (v/v) alcohol, dried, and then resuspended in TE buffer. The resulting total DNA was stored at 4°C for future use.

3) Construction of genomic library

The total DNA of the above-mentioned Pseudomonas fluorescens strain was digested with Sau3A I, and a DNA fragment of about 10 kb was recovered through agarose gel for subsequent use. This fragment was terminally dephosphorylated and ligated into the pACYC184 plasmid vector which was also dephosphorylated. The above ligation product was transferred into Escherichia coli (Escherichia coli) DH5α electroshock competent cells, coated with LB solid medium, and cultured at 37°C for 24 hours for plasmid extraction. In this way, a genomic library containing the gene of interest is constructed.

4) Sequence determination

Pseudomonas fluorescens colonies that grew well on M9 solid basic medium with 0.01wt% tetrachlorobiphenyl as the only carbon source were inoculated in LB liquid medium for overnight culture, and sequenced after plasmid extraction.

5) Sequence analysis

Through nucleotide sequence determination and analysis, the biphenyl degradation-related gene operon fragment is obtained, which has base information such as SEQ ID No 1.

6) Construction of expression vector

The sequence of the biphenyl degradation-related gene operon fragment SEQ ID No 1 was directly connected to the expression vector pMD-18 vector (takara Dalian), connected at 4°C for 12 hours, and then the vector was transformed into Escherichia coli DH5α competent cells (takara Dalian )middle.

After the expression vector containing the fragment of SEQ ID No 1 of the gene operon related to biphenyl degradation is transferred into prokaryotic microbial cells, the cells can synthesize enzymes related to biphenyl degradation. Biphenyl was added to the bacterial solution, and after cultivation, the content of biphenyl decreased.

The beneficial effect that technical solution of the present invention realizes:

The present invention successfully produced a Pseudomonas fluorescens biphenyl degradation-related gene operon. In order to further analyze the role and function of the Pseudomonas fluorescens-derived biphenyl degradation-related gene operon in the degradation of organic pollutants, the analysis The degradation ability of Escherichia coli strains containing this operon to biphenyl was tested. The results show that the biphenyl degradation-related gene operon and its expression vector isolated from Pseudomonas fluorescens can be used to biodegrade biphenyl and polychlorinated biphenyl.

Description of drawings

Figure 1 is the Sau3AI digested fragment, where A is the Sau3AI digested fragment of Pseudomonas fluorescens total DNA.

Detailed ways

The technical solution of the present invention is described in further detail below in conjunction with specific examples, but is not limited to the present invention.

Escherichia coli (Escherichia coli) DH5α strain was preserved by the Plant Genetic Engineering Laboratory, Institute of Biotechnology, Shanghai Academy of Agricultural Sciences. Vectors, various restriction endonucleases, Taq polymerase, ligase, dNTP, 10×PCR buffer and DNA marker were purchased from Bao Bioengineering Dalian Co., Ltd. and New England Biolabs, USA. All chemical reagents were purchased from Sigma Chemical Company, USA and Sinopharm Chemical Reagent Company, Shanghai. ABI PRIAM Big-Dye Terminator DNA sequencing kit was purchased from Applied Systems, Inc.

If there is no special explanation for the molecular biology experiments involved in the present invention, all refer to the book "Molecular Cloning" [Sambrook J, Frets E F, Mannsdes T et al. In: Molecular Cloning. 2nd ed. Cold Spring Harbor Laboratory Press, 1989]. This book and its subsequent publications are the most commonly used guiding reference books for those skilled in the art when performing experimental operations related to molecular biology.

Example 1

Screening of strains degrading polychlorinated biphenyls

(1) Test method:

Take a soil sample [the location is 10 meters of small river silt near an abandoned chemical plant in Laogang, Pudong New Area (formerly Nanhui District), Shanghai], put the soil sample into a sterilized paper bag; weigh 10 g of soil on the ultra-clean workbench Put the sample into a ceramic mortar; add 50mL of sterile water and mash it with a sterile pestle; let it stand for 5min; absorb 1-5mL of the supernatant and add it to 100mL containing 0.01wt% trichlorobiphenyl as the only carbon source cultured at 150 rpm at 28°C for 3 days; 1 mL of the sample was added to a new 100 mL M9 liquid basic medium containing 0.01 wt% trichlorobiphenyl as the sole carbon source to continue subculture. Subculture 4 times in this way; take 20 μL of the plate on the M9 solid basic medium containing 0.01wt% trichlorobiphenyl as the only carbon source.

(2) Test results:

After culturing at 28°C for 3 days, a well-growing Pseudomonas fluorescens strain was obtained.

Example 2

Extraction of total DNA from polychlorinated biphenyl-degrading Pseudomonas fluorescens

(1) Test method:

Inoculate the isolated Pseudomonas fluorescens strain in 100mL liquid LB medium; cultivate overnight (16h) at 28°C and 150 rpm; centrifuge the bacterial culture solution at 6,000g for 5min to obtain bacterial pellet; bacterial pellet Freeze at -20°C for 1h; wash once with TE (10mM Tris-HCl, 1mM EDTA, pH 8.0) solution; add sterile water containing 10mg/mL lysozyme to suspend; incubate at 37°C for 1h; Add 0.5M EDTA, 10% (w/v) SDS and 5M NaCl and gently shake and mix; then add 20mg/mL protein to stimulate K; and incubate the reaction at 37°C for 1h. Use phenol:chloroform:isoamyl alcohol (25:24:1) equivalent to the volume of the culture liquid (1 volume) to extract DNA; add chloroform:isoamyl alcohol (24:1) that is 0.5 times the volume of the equivalent aqueous phase ; Centrifuge for 5 minutes after shaking and mixing; absorb the water phase; add isoamyl alcohol equivalent to the volume of the water phase (1 times the volume); shake and mix; centrifuge for 15 minutes; take the precipitate; wash the DNA with 70% (v/v) alcohol; Dry; resuspend in 50uL TE buffer; store the resulting total DNA at 4°C for future use.

(2) Test results:

The Pseudomonas fluorescens strain isolated by inoculation grew well in 100mL liquid LB medium overnight at 28°C.

Example 3

Construction of genome library of polychlorinated biphenyl-degrading strains

(1) Test method:

Take 10 μL DNA of Pseudomonas fluorescens strain, add 1 μL of Sau3A I enzyme diluted 1:100; digest at 37°C for 10 min, 20 min, 30 min, 40 min, 50 min and 60 min respectively; add 1 μL of 10×loadingbuffer to terminate the reaction; Enzyme digestion reaction time; then select the same system for 30min digestion for a large number of enzymes; 0.7% (w/v) agarose gel electrophoresis; recover 5-8kb DNA fragments. The recovered fragments were recovered using the DNA Agarose Gel Recovery Kit of Hangzhou Weitejie Biochemical Technology Co., Ltd.; after the plasmid vector pACYC184 was completely digested with BamH I, the terminal was dephosphorylated by SAP alkaline phosphatase to reduce the self-ligation of the carrier; the above recovery The final Pseudomonas fluorescens strain DNA (200ng) and the end-dephosphorylated plasmid vector pACYC184 (150ng) were ligated with 2U of T4ligase; ligated at 4°C for 16h; v) and centrifuged with ethanol; finally dissolved with 10 μL of ultrapure water.

(2) Test results:

0.7% (w/v) agarose gel electrophoresis was used to detect the Sau3A I enzymatic hydrolysis of the DNA of the isolated Pseudomonas fluorescens strain, and it was found that the DNA fragments digested at 37°C for 20 min were in a diffuse state (see Figure 1).

Example 4

Transformation of Escherichia coli by electroporation

(1) Test method:

Inoculate a single colony of Pseudomonas fluorescens that grew well on the solid medium and put it into 250mL SOB liquid medium for culture; cultivate overnight at 30°C and 275 rpm; when it grows to OD ₅₅₀ to 0.75, pour the bacterial solution into pre-cool In a centrifuge tube, place on ice for 10 minutes; centrifuge at 5,500 rpm for 5-6 minutes at 4°C, and pour out the supernatant; add sterile water to resuspend the bacteria; centrifuge at 5,500 rpm for 5-6 minutes at 4°C, and pour out the supernatant; Wash again with sterile water, centrifuge at 5500 rpm at 4°C for 5-6min, pour off the supernatant; add 10% sterile glycerol to resuspend the bacteria; centrifuge at 5500 rpm at 4°C for 5-6min, pour off the supernatant ; Use a 1mL pipette tip to blow up the concentrated bacterial solution, which is the electric shock competent cell; take a small amount of DNA (0.1-1ng) sample in 80-100μL competent cells, mix well, and put it on ice for 2min; add the mixture to a pre-cooled 1mm In the electric shock cup, electric shock was performed, and the electric shock parameters were: electric pulse 2.5 μF, voltage 2.5 kV, resistance 200 Ω, electric shock time 4.5 s); Immediately add 1 mL of culture medium, shake and incubate at 37 ° C for 1 hour.

Spread the bacterial solution on an LB (containing 50 μg/mL ampicillin) plate, and incubate at 37°C for 12-16 hours;

Extract the plasmid and store at -20°C.

(2) Test results:

The above ligation product was electroporated into Escherichia coli DH5α electroporation-competent cells, coated with LB solid medium, cultured at 37°C for 24 hours, and the transformant bank capacity was 4×10 ⁴ . A large-scale extraction of plasmids was carried out to construct a genomic library containing the target gene.

Example 5

Screening of transformants that degrade PCBs

(1) Test method:

The genome library of the PCB-degrading strain was transformed into Escherichia coli DH5α; the plate was placed on M9 solid basic medium containing 0.01 wt% trichlorobiphenyl as the sole carbon source; cultured at 28°C for 3 days.

(2) Test results:

E. coli colonies grew well.

Example 6

Sequence Determination and Analysis of Biphenyl Degradation-Related Gene Operators

(1) Test method:

Introduce well-growing E. coli colonies into 2 mL of LB medium for overnight culture; shake at 37 degrees (150 rpm) for overnight culture. Take 1.5ml, centrifuge at 13000rpm for 30 seconds; pour off the supernatant, suspend the bacteria in 100ml pre-cooled solution I, Vortex. Add 200ul freshly prepared solution II, and mix up and down quickly. Add 150ul pre-cooled solution III, mix up and down quickly, and place on ice for 15 minutes. Centrifuge at 13000 rpm at 4°C for 15 minutes. Transfer the supernatant to another centrifuge tube, add 900ul pre-cooled absolute ethanol, and mix well. Leave on ice for 10 minutes. Centrifuge at 13000rpm at 4°C for 15 minutes. Pour off the supernatant, add 1ml of 70% ethanol, and wash the DNA pellet. Centrifuge at 13000rpm for 5 minutes, and remove the supernatant. Add 30 μl TER to dissolve the precipitate, and after 30 minutes in 37°C water bath, store at -20°C until use. Sequencing of the extracted plasmids was performed.

(2) Test results:

After electrophoresis detection of E. coli plasmid, the plasmid content was about 100ng/μl. DNA sequencing showed that the operon of biphenyl degradation-related genes was 6716bp in length, including 1313 bases A, 1955 bases C, 2117 bases G, and 1331 bases T. See sequence SEQ NO 1 for details.

Example 7

HPLC detection of biphenyl degradation

(1) Test method:

Add 100mL LB culture solution to a 250mL Erlenmeyer flask, insert 100μL of Pseudomonas fluorescens strain; culture overnight at 28°C, 120 rpm; add 1mL prepared biphenyl solution (50mg/mL, 1g biphenyl dissolved in 20mL methanol); continue to culture at 28°C, 120 rpm; after 3 days, pipette 500μL of the bacterial solution into the Dorf tube, and then add 500uL of ether; oscillate for 5-10min to mix well; Add 500 μL methanol to the Dorf tube, mix well and then rotatively evaporate; suck out the remaining methanol, then add 500 μL methanol; filter the membrane into a new Dorf tube; put on the column, HPLC conditions: the machine is Agilent 1100 series, Mobile phase (acetonitrile:water=70:30), flow rate 1.0mL/min, stationary phase (C18 reversed column), column temperature (normal temperature), detection wavelength (254nm), injection volume (20μL).

(2) Test results:

The concentration of pure biphenyl was 100 μg/mL, and the peak eluting time was about 5.9 minutes. After 1 day, the Escherichia coli containing the biphenyl degradation-related gene operon degraded about 46.8% of the biphenyl in the medium, while the control blank degraded 1.5%, and the blank E. coli degraded about 3.2%. The results indicated that the Escherichia coli containing the biphenyl degradation-related gene operon of the present invention had obvious ability to degrade biphenyl (see Table 1).

Table 1 HPLC detection of biphenyl degradation analysis

                 

No. Degradation Efficiency

                 

Blank control 1.5%

Blank Escherichia coli strain 3.2%

Escherichia coli containing the gene operon related to biphenyl degradation of the present invention 46.8%

                 

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation. Although the present invention has been described in detail with reference to preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the invention can be Modifications or equivalent replacements without departing from the spirit and scope of the technical solutions of the present invention shall be covered by the claims of the present invention.

Claims (7)

1. a biphenyl degraded genes involved operon that comes from Pseudomonas fluorescens is characterized in that the base sequence of said genetically manipulated is shown in SEQ ID No 1.

2. genetically manipulated according to claim 1 is characterized in that said genetically manipulated contains 1313 of base A, 1955 of base C, 2117 of bases G, 1331 of base T.

3. claim 1 or 2 application of described genetically manipulated in biological degradation biphenyl and polychlorobiphenyl.

4. claim 1 or the 2 described genetically manipulated application in prokaryotic organism degraded biphenyl and polychlorobiphenyl.

5. one kind comprises claim 1 or the 2 described expression vectors that come from the biphenyl degraded genes involved operon of Pseudomonas fluorescens.

6. expression vector according to claim 5 is characterized in that, described expression vector is the TA carrier, and it is through directly being connected to form with SEQ ID No 1 sequence.

7. claim 5 or 6 application of described expression vector in biological degradation biphenyl and polychlorobiphenyl.