Citrobacter freundii with transformed phosphorus accumulating genes and construction method and application thereof

A technology of Freund's citric acid and polyphosphate kinase, which is applied in the field of genetic engineering, can solve problems such as environmental application to be discussed, and achieve the effect of improving the removal capacity

Active Publication Date: 2015-04-22
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Since Ppk (belonging to the Ppk1 family) was first discovered in Escherichia coli (Escherichia coli, E.coli), its related coding gene has been cloned, and the Ppk1 protein translated into it has been purified. Genetic methods are used to achieve bioaugmented phosphorus removal. At present, there have been reports that the Ppk1 gene of E.coli has been expressed in E.coli and achieved remarkable results. However, in view of the particularity of E.coli identity, its environmental application remains to be discussed.

Method used

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  • Citrobacter freundii with transformed phosphorus accumulating genes and construction method and application thereof
  • Citrobacter freundii with transformed phosphorus accumulating genes and construction method and application thereof
  • Citrobacter freundii with transformed phosphorus accumulating genes and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Construction of Citrobacter freundii transgenic for phosphorus accumulation gene.

[0057] (1) PCR amplification of Citrobacter freundii Ppk1 gene:

[0058] According to the Citrobacter freundii ATCC 8090 "whole genome shotgun sequence" provided in the NCBI database, GenBank accession number: ANAV01000007.1, design two primers - forward primer (named CFPPKF): 5'-GGGGTACCAatgggtcaggaaaagctatacatcg-3', reverse primer (named CFPPKR): 5'-CCCAAGCTTttagtcaggttgctcgagtgatttg-3', using Citrobacter freundii ATCC 8090 genomic DNA as a template, PCR amplification, amplification conditions: 95°C for 5min, 98°C for 10Sec, 68°C for 2min, 30 cycles, 72°C for 5min.

[0059] (2) Sequence analysis of Citrobacter freundii Ppk1 gene:

[0060] The PCR product was cleaned and recovered for adding A reaction, and after cleaning and recovery again, it was ligated with the T-Vector pMD19 (Simple) vector to obtain the T-Ppk1 plasmid, which was transformed into E.coli DH5α competent ...

Embodiment 2

[0068] Example 2: PCR amplification results of Citrobacter freundii Ppk1 gene.

[0069] According to the Citrobacter freundii ATCC 8090 (whole genome shotgun sequence) GenBank accession number provided in the NCBI database: the primers designed by ANAV01000007.1 amplified a Ppk1 gene fragment with a size of 2067 bp from Citrobacter freundii ATCC 8090 genomic DNA ( See sequence listing SEQ ID NO.1) for detailed sequence, such as figure 1 As shown, the position of the band is correct. At the same time, the sequence result was compared with the sequence reported in the NCBI database using Bio-Edit software, and the base sequence was also completely consistent.

Embodiment 3

[0070] Example 3: Construction results of Citrobacter freundii Ppk1 gene expression vector.

[0071] The full length of the broad host expression vector pBBR1MCS-2 is 5144bp (see the sequence listing SEQ ID NO.2 for the detailed sequence), such as figure 2As shown, after inserting the 2067bp Ppk1 gene fragment of Citrobacter freundii into the multiple cloning site of the expression vector, the vector became significantly larger, and the sequence was completely correct after double enzyme digestion and sequencing comparison.

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Abstract

The invention discloses a Citrobacter freundii with transformed phosphorus accumulating genes. Polyphosphate kinase Ppk1 genes derived from the Citrobacter freundii are led into the Citrobacter freundii. The DNA of the Citrobacter freundii only contains Ppk1 genes, and the control way of the Ppk1 genes and Ppx genes is dual-cis-trans cotranscription. The phosphorus accumulation ability of bacteria with the characteristics can be improved by means of the Ppk1 with host bacteria as a receptor to express the source. The invention further discloses a construction method of the Citrobacter freundii with the transformed phosphorus accumulating genes and the application of the Citrobacter freundii with the transformed phosphorus accumulating genes in waste water dephosphorization. The Citrobacter freundii with the transformed phosphorus accumulating genes has the advantage of being high in dephosphorization ability.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a strain of Citrobacter freundii transmuted with a phosphorus-accumulating gene and its construction method and application. Background technique [0002] In freshwater bodies, controlling or alleviating eutrophication largely depends on controlling the input of phosphorus nutrients. The sources of phosphorus-containing nutrients input into water bodies are various, including natural processes and human activities, but the main reason that causes excessive phosphorus in water bodies and leads to eutrophication of water bodies is mainly human activities. In my country, the main sources of phosphorus-containing pollutants are domestic pollution sources, agricultural pollution sources, livestock and poultry pollution sources, and industrial pollution sources. In order to solve the harm caused by phosphorus pollution, it is necessary to effectively treat the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C02F3/34C12R1/01
CPCC02F3/342C02F2101/105C12N9/1229C12N15/74C12Y207/04001
Inventor 杨柳燕王鑫陈旭张文李丽王爱丽吴丹
Owner NANJING UNIV
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