Bacillus megaterium co-expression vector for nitrite reductase and construction method thereof

A technology of Bacillus megaterium and co-expression vector, applied in the field of genetic engineering, can solve the problems of failing to meet the requirements of repairing the environment and low expression of nitrite reductase

Inactive Publication Date: 2014-03-05
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, generally speaking, the expression level of nitrite reductase in microorganisms is very low, which cannot meet the requirements of repairing the environment. Ther

Method used

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  • Bacillus megaterium co-expression vector for nitrite reductase and construction method thereof
  • Bacillus megaterium co-expression vector for nitrite reductase and construction method thereof
  • Bacillus megaterium co-expression vector for nitrite reductase and construction method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Cultivation of Bacillus megaterium NCT-2

[0020] Bacillus megaterium NCT-2 was isolated from a greenhouse cultivated in Chongming Island, Shanghai for 12-15 years. The preservation number is CGMCC NO.4698. No. 3, No. 1 Yard, Beichen West Road, District, Institute of Microbiology, China; date of preservation is March 21, 2011. The bacteria were inoculated in LB liquid medium and cultured at 32°C for OD 600 To about 1.5, the bacteria were collected by centrifugation and used to extract the genomic DNA of the bacteria.

[0021] The components of the LB liquid medium are: 10 g of peptone, 5 g of yeast extract, 10 g of NaCl, 1 L of deionized water, and pH 6.8-7.2. LB solid medium is 15-20g agar added on the basis of LB liquid medium.

Embodiment 2

[0023] Construction of pETDuet-1-Bm-Nas D recombinant vector

[0024] According to the nitrite reductase electron transfer subunit (Bm-Nas D) gene sequence obtained by sequencing, corresponding primers were designed to amplify the gene and respectively connected to the pETDuet-1 co-expression vector.

[0025] Primers were designed as follows:

[0026] Forward primer Nas D-BamH I-F:

[0027] 5'-CGGGATCCATGCAAAAAAAGAAACTCGTATTGAT-3'

[0028] Reverse primer Nas D-EcoR I-R:

[0029] 5'-CGGAATTCTTATGATGTGACAACTGTTTCGAACAG-3'

[0030] BamH I and EcoR I restriction sites were designed at the 5' ends of the upstream and downstream primers to connect to the co-expression vector pETDuet-1, and the gene sequence of the large subunit of nitrite reductase was amplified with the primers containing the restriction sites. The PCR product was ligated to the pMD19-T (Simple) vector, and the ligated product was transformed into DH5α Escherichia coli competent cells. The positive clones were...

Embodiment 3

[0033] Construction of pETDuet-1-Bm-Nas D-Nas E recombinant vector

[0034] According to the nitrite reductase small subunit (Bm-Nas E) gene sequence obtained by sequencing, corresponding primers were designed to amplify the gene and connected to the pETDuet-1-Bm-Nas D recombinant vector.

[0035] Primers were designed as follows:

[0036] Forward primer Nas E-Kpn I-F:

[0037] 5'-GGGGTACCATGGTGAATAAAAACATTACAAAAG-3'

[0038] Reverse primer Nas E-Xho I-R:

[0039] 5'-CCGCTCGAGTTATTCCTCAATTAAAAGATACAGCA-3'

[0040] Use primers containing Kpn I and Xho I restriction sites to amplify the nitrite reductase small subunit gene sequence and connect the PCR product to the pMD19-T (Simple) vector, and transfer the ligated product into DH5α Escherichia coli competent cells . The positive clone was picked and its plasmid was recovered, and then digested with Kpn I and Xho I to recover the fragment Bm-Nas E' containing the catalytic subunit of nitrite reductase. Digest the pETDuet-1...

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Abstract

The invention relates to a bacillus megaterium co-expression vector for nitrite reductase and a construction method thereof, belonging to the technical field of genetic engineering. According to the co-expression vector, all genome sequences of bacillus megaterium are determined and serve as a template, gene sequences containing restriction enzyme cutting sites, namely a large subunit (Bm-Nas D) and a small subunit (Bm-Nas E) of nitrite reductase, are amplified according to designed PCR (Polymerase Chain Reaction) primers and are sequentially connected to the co-expression vector pETDuet-1, and positive clones of Escherichia coli DH5alpha containing target gene fragments are selected by a colony PCR technology. The co-expression vector has the advantages that the defect in the prior art that the use effect is seriously restricted due to the fact that the expression level of the gene sequence of the small subunit of nitrite reductase in natural bacillus megaterium is very low is overcome, and the genetic expression level is increased greatly.

Description

technical field [0001] The present invention relates to a method in the technical field of genetic engineering, specifically a nitrite reductase small subunit gene sequence, a recombinant vector containing the gene, a recombinant bacterium, and the expression product of the gene produced by the recombinant bacterium and its application. Background technique [0002] From an environmental point of view, the accumulation of N elements can cause waste of fertilizers and affect crop growth and soil utilization. At the same time, it can cause agricultural non-point source pollution through farmland surface runoff and farmland leakage; from the perspective of food safety, the human body Nitrate (NO 3 –) easily reduced to nitrite (NO 2 –), nitrite can oxidize the oxygen-carrying low iron hemoglobin in the cells to metherin without oxygen-carrying capacity and cause tissue hypoxia. Not only that, the oxidized methemoglobin will also reduce the oxygenation of the oxygen-carrying T...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/66C12N1/21B09C1/10C12R1/11
Inventor 周培冯海玮时唯伟支月娥刘群录陆伟初少华孙玉静卫星
Owner SHANGHAI JIAO TONG UNIV
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