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Recombinant pseudomonas putida, and construction method and application thereof

A technology of Pseudomonas putida and a construction method, applied in the field of recombinant Pseudomonas putida and its construction, and can solve the problems of unrealistic phosphorus removal and the like

Inactive Publication Date: 2010-12-01
NANJING UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is impractical to use E.coli directly for phosphorus removal in wastewater or natural water

Method used

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  • Recombinant pseudomonas putida, and construction method and application thereof
  • Recombinant pseudomonas putida, and construction method and application thereof
  • Recombinant pseudomonas putida, and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Acquisition of ppk gene.

[0034] The total DNA of E.coli DH5α was extracted. For specific methods, refer to "Environmental Microbial Experimental Technology" (Beijing: China Environmental Science Press, 2004.71-72). Using the E.coli DH5a genome as a template, primers were designed according to the DNA sequence of the ppk gene (Genebank accession number: L03719), and the ppk gene was amplified by PCR for transgenic operation.

[0035] Forward primer: 5′-ATG CAG ATG GGT CAG GAA AAG CTA TAC AT-3′;

[0036] Reverse primer: 5'-GGT ACC CAG GTT GTT CGA GTG ATT TGA-3'.

[0037] The PCR reaction conditions were: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 60°C for 45 s, extension at 72°C for 2 min, and the number of cycles was 30.

[0038] The enzyme used in PCR was PrimeSTAR purchased from TAKARA (JAPAN) TM HS DNA polymerase. A fragment of about 2 kb was amplified. The enzymes used in PCR are high-fidelity enzymes and the prod...

Embodiment 2

[0039] Example 2: Construction of vectors.

[0040] The PCR product obtained in Example 1 was subjected to a phosphorylation reaction, and phosphorus was added to the 5' end. Plasmid pBBR1MCS-2 [Kovach, M., P. Elzer, et al. (1995). "Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes." Gene 166(1):175-176.] After EcoR V digestion, dephosphorylation reaction was performed to avoid self-ligation. The PCR product (ppk) treated above was reacted overnight with plasmid pBR1MCS-2. Single clones were picked for sequencing. The recombinant plasmid pBR1MCS-2-ppk inserted in the forward direction was obtained. Transformed into the host E.coli DH5α by electroporation for replication. The recombinant plasmid pBBR1MCS-2-ppk was extracted and used as a template to design primers containing Not I restriction sites.

[0041] Forward primer: 5′ GCG GCC GC C GTT GCG TCG CGG TGC A-3' (Not I);

[0042] Reverse primer: ...

Embodiment 3

[0046] Example 3: Triparental conjugation.

[0047] The recombinant plasmid pUTmini-Tn5-ppk was transformed into the donor strain E.coli SM10(λpir) by electric shock (for the preparation of electric shock transformation and competent cells, please refer to the "Molecular Cloning Guide" [3 rd , Beijing: Science Press. 2002.]).

[0048] The recipient bacterium (Pseudomonas putida KT2440), the donor bacterium [E.coli SM10(λpir) / pUTmini-Tn5-ppk] containing the corresponding plasmid and the auxiliary bacterium E.coli DH5α(pRK600) were cultured overnight in LB medium. And add chloramphenicol (final concentration is 25 μg / mL) in KT2440 medium, add the final concentration of 10 μg / mL kanamycin and 25 μg / mL chloramphenicol in the culture medium of donor bacteria and auxiliary bacteria . The recipient bacteria were incubated at 42°C for 15 minutes to inactivate the restriction system. Then add 2 mL of donor bacteria and 2 mL of auxiliary bacteria to 1 mL of recipient bacteria and mix...

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Abstract

The invention discloses recombinant pseudomonas putida, which is expressed on the basis of DNA integration. The invention also discloses a construction method for the recombinant pseudomonas putida, and the application of the recombinant pseudomonas putida in phosphorus removal from wastewater. The method of the invention is simple, and obtained genetically engineered bacteria have high-efficiency phosphorus removal capacity.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant Pseudomonas putida and its construction method and application. Phosphorus in domestic sewage can be removed by using the recombinant Pseudomonas putida. Background technique [0002] Inorganic phosphate is generally considered to be a key factor in lake eutrophication. Therefore, reducing the concentration of phosphorus in the water environment is one of the important means to control eutrophication. [0003] Phosphorus can be removed from the environment by physical, chemical and biological methods. The mechanism of phosphorus removal by organisms, especially microorganisms, is that bacteria including Escherichia coli and Pseudomonas spp. have the ability to accumulate inorganic phosphate and generate polyphosphate (poly-phosphate). However, under natural conditions, the ability of microorganisms to accumulate phosphorus is usually weak, ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/54C12N15/63C02F1/58C02F3/34C12R1/40C02F101/10
Inventor 武俊杜宏伟杨柳燕肖琳李丽观吕志刚陈洪龄凌小君张全兴
Owner NANJING UNIV
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