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Hexavalent chromium reductive gene in bacillus thuringiensis YB-03, expression product and application of hexavalent chromium reduction gene

A technology of YB-03 and Bacillus aureus, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of long reduction time, low reduction ability of hexavalent chromium, low resistance of hexavalent chromium, etc., and achieve strong reduction sexual effect

Active Publication Date: 2012-09-19
HENAN ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] These existing microorganisms have low resistance to hexavalent chromium, or low reduction ability of hexavalent chromium, or require a long reduction time, and generally have a certain distance from industrial application

Method used

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  • Hexavalent chromium reductive gene in bacillus thuringiensis YB-03, expression product and application of hexavalent chromium reduction gene
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  • Hexavalent chromium reductive gene in bacillus thuringiensis YB-03, expression product and application of hexavalent chromium reduction gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Isolation and screening of hexavalent chromium-reducing bacteria

[0025] Take 10g of activated sludge (collection time: September 2010; collection location: Zhengzhou Wulongkou Sewage Treatment Plant; collection person: Wei Fei) in 90ml of sterile water, and dilute to 10 -1 ~10 -6 Six gradients. Take 1ml of the solution from each gradient and spread it on 6+ LB solid medium, cultured at 37°C for 24h.

[0026] Reductive identification of chromium-resistant bacteria: select colonies from the culture medium containing 100mg / L Cr 6+ In LB liquid medium, cultured at 37°C, 150r / min for 24h, took out an appropriate amount of solution and centrifuged to get the supernatant, and measured the remaining Cr in the solution 6+ content.

[0027] After preliminary screening in this collection, two strains with different shapes were obtained, which were named YB-01 and YB-03 respectively. The reducing effects of the two strains on hexavalent chromium are shown in Table...

Embodiment 2

[0032] Embodiment 2 The PCR of hexavalent chromium reduction gene, connection with PMD19-T, transformation, identification

[0033] (1) Design of primers

[0034] The Primer 5 software was used to design the primers as follows:

[0035] CR- EcoRI-F: gcgaattcATGAATGAGATGATACAT (as shown in SEQ ID NO.3)

[0036] CR-XholⅠ-R: gcctcgagTCACTTCTTATTAAATCC (as shown in SEQ ID NO.4)

[0037] PCR system (50ul): Mix: 25ul; upper primer: 1ul; lower primer: 1ul; dd H 2 O 22ul;( Total DNA of YB-03 bacteria ) DNA template: 1ul;

[0038] First, pre-denaturation at 94°C for 5 minutes, and then into a cycle: DNA denaturation at 94°C for 40 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 70 seconds, 30 cycles, and finally extension at 72°C for 7 minutes.

[0039] (2) Electrophoresis and gel cutting recovery of PCR products

[0040] After the PCR product was electrophoresed on 1% agarose, soaked in EB for 30min, the result was observed in the gel imaging system. Afte...

Embodiment 3

[0057] Example 3 Connection, transformation and identification of hexavalent chromium reduction gene and PET-28

[0058] (1) Ligate the hexavalent chromium target gene recovered from the gel with PMD19-T, transform it into TG1 competent to obtain a positive clone, shake the bacteria overnight, extract the plasmid, and double enzyme digest to obtain the target gene.

[0059] PMD19-T+ target gene plasmid double-enzyme electrophoresis results see figure 2 .

[0060] Depend on figure 2 It can be seen that two bands appeared after double enzyme digestion, one of which was above 2500bp, which was the PMD19-T fragment, and the other was the target fragment, and the size of the band was about 750bp.

[0061] (2) After recovering the target fragment at 750 bp, connect it to the PET-28 carrier, transform it into BL21(DE3) competent, and obtain a positive clone. After overnight shaking, the plasmid is extracted and verified by double enzyme digestion.

[0062] The PET-28 vector + ta...

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Abstract

The invention relates to hexavalent chromium reductive gene in bacillus thuringiensis YB-03, an expression product and application of the hexavalent chromium reduction gene. According to the invention, the hexavalent chromium reductive gene is cloned from bacillus thuringiensis YB-03 strain CGMCC NO.5653 with the action of restoring Cr<6+>, and high-activity protease is successfully transformed and induced to express. The protease has strong reducibility to hexavalent chromium, and has important application value in treatment of chromium pollution.

Description

technical field [0001] The invention relates to a chromium-resistant bacteria gene, in particular to a hexavalent chromium reduction gene in bacillus thuringiensis YB-03, its expression product and its application. Background technique [0002] Although chromium is an essential trace element for the human body, excessive chromium, especially hexavalent chromium, is highly toxic and highly pathogenic to the human body. The annual output of chromium salt in my country has exceeded 160,000 tons, while the discharge of chromium slag is 350,000 to 420,000 tons, which contains about 3,500 tons of hexavalent chromium. Hexavalent chromium is extremely toxic and is one of the main heavy metals causing environmental pollution. Therefore, the control of chromium pollution has aroused widespread concern. Traditional physical and chemical treatment methods include pH adjustment, flocculation precipitation, ion exchange and activated carbon adsorption, etc., but these methods need to co...

Claims

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Application Information

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IPC IPC(8): C12N15/57C12N9/54C12N15/63C12N1/21C12N15/11C12R1/07
Inventor 薛保国魏斐杨丽荣
Owner HENAN ACAD OF AGRI SCI
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