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Acidithiobacillus ferrooxidans genetic engineering bacteria and use thereof

A technology of Thiobacillus ferrooxidans and genetically engineered bacteria, which is applied in the direction of bacteria, chemical instruments and methods, and process efficiency improvement, and can solve the problems of improving acidophilic Thiobacillus ferrooxidans, etc.

Inactive Publication Date: 2011-01-05
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After searching, there is currently no information on how to increase the Fe content of Acidophilus ferrooxidans in a transgenic way. 2+ report on the oxidation capacity of ions

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Construction and identification of recombinant plasmid pTCYC1.

[0048] (1) After linking the cyc1 gene and the 6×His tag encoding gene with the shuttle plasmid pJRD215, transform Escherichia coli DH5α, screen on the LB solid plate containing Km to obtain Escherichia coli DH5α containing the Km resistance plasmid, and extract the plasmid enzyme It was verified by cutting that the plasmid carried by the transformant indeed contained the insert.

[0049] (2) After linking the Ptac gene fragment with the intermediate vector pCYC1, transform Escherichia coli DH5α, screen the LB solid plate containing Km to obtain Escherichia coli DH5α containing the Km-resistant plasmid, and verify the enzyme digestion of the extracted plasmid. Plasmids do contain inserts. The verified positive clones were sequenced, the clones with correct sequencing were selected, the plasmids were extracted, and transformed into Escherichia coli DH5α (RP4) and screened on the LB solid plate c...

Embodiment 2

[0051] Example 2: Construction and identification of acidophilus Thiobacillus ferrooxidans genetic engineering bacteria.

[0052] (1) Escherichia coli DH5α (RP4 / pTCYC1) in the mid-exponential growth phase was collected as the donor bacteria, and acidophilic Thiobacillus ferrooxidans ATCC19859 in the mid-exponential growth phase was collected as the recipient bacteria. They were washed 3 times with inorganic salt washing solution, and the same bacteria The body concentration was suspended in the washing solution for later use.

[0053] (2) According to the volume ratio of the donor bacteria and the acceptor bacteria as 1:1, mix the suspension, take 200 μl and put it on a nitrocellulose filter membrane with a pore size of 0.45 μm (the smooth side is facing upwards), at 30°C Cultivate on the junction plate for 48 hours; 1ml of washing solution washes down the bacteria on the filter membrane and dilutes to 10 -1 、10 -2 、10 -3 、10 -4 , 100 μl were taken to coat a 2:2 screening ...

Embodiment 3

[0057] Example 3: Expression of the cyc1 gene carried by the recombinant plasmid pTCYC1 in acidophilic Thiobacillus ferrooxidans

[0058] (1) Strain selection: A. ferrooxidans pTCYC1, A. ferrooxidans ATCC19859;

[0059] (2) Seed culture: put A.ferrooxidans pTCYC 1 and A.ferrooxidans ATCC19859 in 30mL 9K-FeSO 4In the liquid medium, under the condition of 30°C, shake culture until the stationary phase, and obtain the seed liquid;

[0060] (3) Expansion cultivation: inoculate the seed liquid of two strains of bacteria in 200mL9K-FeSO4 with the inoculation amount of 5% volume ratio 4 In the liquid medium, under the condition of 30°C, culture with shaking until the stationary phase (about 4d);

[0061] (4) SDS-PAGE electrophoresis of acidic Thiobacillus ferrooxidans total protein: filter the culture solution obtained in step (3) to remove high-iron precipitates, collect the bacteria by centrifugation, resuspend the bacteria in lysis buffer, and ultrasonically disrupt For cells, ...

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PUM

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Abstract

The invention discloses Acidithiobacillus ferrooxidans genetic engineering bacteria, which are A. ferrooxidans pTCYC1 with high ferrous oxidization activity, and are characterized in that: the bacteria are named Acidithiobacillus ferrooxidans, and the strain was collected in China General Microbiological Culture Collection Center with a collection number of CGMCC No.3549 on December 28th, 2009. The genetic engineering bacteria of the invention have higher ferrous oxidization activity and more stable performance than wild thiobacillus ferrooxidans and have a promising application prospect in fields of biological leaching, smoke desulfuration, acid mine water processing, sludge heavy metal removal and the like.

Description

technical field [0001] The present invention relates to a strain of Thiobacillus ferrooxidans and its application, in particular to a strain of genetically engineered acidophilic Thiobacillus ferrooxidans with high ferrous oxidation ability and its construction and application. Background technique [0002] Acidithiobacillus ferrooxidans is one of the species that plays a key role in the bacterial leaching process. acidophilus Thiobacillus ferrooxidans in Fe 2+ When grown in a substrate, Fe 2+ is rapidly oxidized to Fe 3+ provide electrons to enter the bacterial respiratory chain, and Fe 3+ As a strong oxidizing agent, it plays a key role in processes such as bioleaching. Thus, Acidophilus ferrooxidans oxidizes Fe 2+ The ability of ions is an important indicator to measure its leaching ability. After searching, there is currently no information on how to increase the Fe content of Acidophilus ferrooxidans in a transgenic way. 2+ A report on the oxidation capacity of i...

Claims

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Application Information

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IPC IPC(8): C12N1/20C22B3/18B01D53/84B01D53/48C02F11/02C02F3/34C12R1/01
CPCY02A50/20Y02P10/20
Inventor 林建群刘玮林建强刘相梅颜望明
Owner SHANDONG UNIV
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