Construction method for polyphosphate kinase gene transformed Escherichia coli

A technology of Escherichia coli and construction method, which is applied in the field of constructing Escherichia coli transfected with polyphosphokinase gene, can solve problems such as biological phosphorus removal, reduce control difficulty, remove phosphorus efficiently, and reduce treatment costs

Inactive Publication Date: 2006-12-13
NANJING UNIV
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  • Abstract
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Problems solved by technology

[0006] At present, there is no relevant report on the study of phosphorus accumulation genes in China. The research on transgenic phosphorus accumulation bacteria abroad mainly focuses on the mechanism of phosphorus metabolism, and there is no report on the application of biological phosphorus removal.

Method used

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  • Construction method for polyphosphate kinase gene transformed Escherichia coli
  • Construction method for polyphosphate kinase gene transformed Escherichia coli
  • Construction method for polyphosphate kinase gene transformed Escherichia coli

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Embodiment Construction

[0044]1. Cloning of ppk gene: E. coli DH5α was cultured overnight in LB medium, and its genomic DNA was extracted according to an existing method. When cloning the ppk gene, use the total DNA of E. coli DH5α as a template, and perform PCR (polymerase chain reaction) amplification with Taq DNA polymerase (Promega, USA). The forward primer sequence for PCR amplification is: 5'-GAATTTCTAGAATGGGTCAGGAAAAG-3', and the reverse primer sequence is: 5'-GCCGTGAGCTCTTATTCAGGTTGTTCGAG-3'. The reaction conditions of PCR were 94° C. for 1 min, 25 cycles (94° C. for 30 s, 58° C. for 45 s, and 72° C. for 2.5 min), and then 72° C. for 10 min. The amplified target gene was recombined into the cloning vector pMD18-T (Takara, Japan), and then transformed into DH5α. The correctness of the target gene sequence was confirmed by sequencing.

[0045] 2, the construction of expression vector: by adopting restriction endonuclease BamH I and HindIII (Takara, Japan) to double digest the pMD18-T plasmid ...

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Abstract

The invention discloses a construction approach for bacillus coli of transpolyphosphokinase gene, in which the said method includes gene-clone and carrier-construction; the clone includes extracting master DNA of bacillus coli; designing primers; augmenting ppk gene; restructuring the augmented object gene into clonic carrier pMD18-T and converting bacillus coli DH5 alpha; the construction includes adopting restriction enzymes of BamHI and HindIII bisenzyme to cut pMD18-T plasmid with ppk object gene and idle expression carrier pET-28a(+); directionally connecting the object gene by T4 to the expression carrier pET-28a(+), then converting the DH5 alpha; by the PCR and bisenzyme pressure methods screening and verifying the positive recon; extracting the recombination plasmid Pet28a-PPK and converting the acceptor strain BL21(DE3). The invention is of simple process and the obtained gene has efficient phosphorous removal ability.

Description

1. Technical field [0001] The invention relates to a method for constructing a phosphokinase-transferred Escherichia coli, which can remove phosphorus in waste water by using the phosphokinase-transferred Escherichia coli. 2. Background technology [0002] Water eutrophication has become a major environmental problem facing our country. Phosphorus is often the key factor controlling lake eutrophication. If phosphorus in water can be effectively removed, the occurrence of eutrophication can be inhibited and the water quality of lakes can be greatly improved. Environmental Quality. [0003] Since many microorganisms have the ability to excessively accumulate phosphorus, the use of microorganisms for biological phosphorus removal has the advantages of low operating costs and less secondary pollution. In the traditional phosphorus removal process, under the condition of aerobic / anaerobic alternation, phosphorus accumulating bacteria become the dominant bacteria group in wastewa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/52C12N15/70C12N15/66C12R1/19
Inventor 杨柳燕王勤赵庆顺肖琳蒋丽娟尹大强任晶
Owner NANJING UNIV
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