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Copper-resistant genetic engineering bacteria of acidithiobacillus ferroxidans and application thereof

A technology of Thiobacillus thiooxidans and genetically engineered bacteria, applied in the direction of bacteria, improvement of process efficiency, and methods based on microorganisms, can solve problems such as environmental hazards, loss, and reduction of biological characteristics of engineering bacteria, and achieve the effect of expanding the scope of application

Active Publication Date: 2014-09-03
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, in Acidithiobacillus sp., researchers only improve or increase some biological characteristics of the genus by introducing recombinant plasmids; however, the genetically engineered bacteria constructed in the form of plasmids have poor stability, Under the pressure of no antibiotics, the plasmids in the engineering bacteria are easy to lose, so that certain biological characteristics in the engineering bacteria are reduced or even lost. Therefore, in order to improve the stability of the plasmids in the engineering bacteria, antibiotics must be added to the environment in which they live; Thiobacillus acidic acid is mainly used in bioleaching, heavy metal pollution control and other fields. Adding antibiotics to the above environment will cause harm to the environment on the one hand, and on the other hand, expensive antibiotics will undoubtedly increase industrial costs, which is not conducive to this kind of genetic engineering. Large-scale application of bacteria

Method used

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  • Copper-resistant genetic engineering bacteria of acidithiobacillus ferroxidans and application thereof
  • Copper-resistant genetic engineering bacteria of acidithiobacillus ferroxidans and application thereof
  • Copper-resistant genetic engineering bacteria of acidithiobacillus ferroxidans and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Anti-copper gene copB Af Integrated plasmid pZK19-copB Af construction and identification of

[0029] 1. Construction of the plasmid backbone pZK19-IngUH-IngDH for A. thiooxidans ATCC19377 gene integration

[0030] On the genome of A.thiooxidans ATCC19377, the spacer region of two genes with opposite transcription directions was selected as the insertion site of the exogenous gene; primers were designed to amplify a fragment about 1.0 kb upstream and downstream of the insertion site as the homologous recombination arm:

[0031] IngUHF: 5'-CTGTCGACGGG GATAGATGACATTC-3'

[0032] IngUHR: 5'-GTAAGCTTGCCTGATCAGCTCACGGT-3'

[0033] IngDHF: 5'-TTATGGATCCGGCTTCAGGCATCGCCGT-3'

[0034] IngDHR: 5'-GGCGCTCTAGACTCTTAATGGTCCTATTCCGAG-3'

[0035] A SalI restriction site was added to the 5' end of the primer IngUHF, a HindIII restriction site was added to the 5' end of the primer IngUHR, a BamHI restriction site was added to the 5' end of the primer IngDHF, and a BamH...

Embodiment 2

[0047] Example 2: Construction and Identification of Acidophilus Thiobacillus Thioxidans Copper-Resistant Genetic Engineering Bacteria

[0048] 1. Build copB Af Gene-integrated A.thiooxidans single-crossover strain

[0049] The mid-log phase Escherichia coli S17-1λpir (pZK19-copB Af ) as the donor bacteria and acidophilic thiothiooxidans ATCC19377 in the late logarithmic phase as the recipient bacteria, and mixed according to the ratio of donor bacteria: acceptor bacteria 1:1.5, spread on the Starkey-Na 2 S 2 o 3 On the 0.22um filter membrane of the junction medium plate, culture at 30°C for 7 days; use Starkey-Na 2 S 2 o 3 Inorganic salt solution to elute the bacteria on the filter membrane, and apply to Starkey-Na 2 S 2 o 3 On a solid plate, culture at 30°C for 12 days; then add 1.0 μL of 12 mg / mL sterile X-gal solution dropwise to the above-mentioned single colony, place it at 30°C for 30 minutes, pick a blue colony for PCR amplification and agarose Gel electropho...

Embodiment 3

[0082] Example 3: A. thiooxidans copB Af (CGMCC No.9286) Application of Genetic Engineering Bacteria in Bioleaching

[0083] 1. A. thiooxidans cop B Af (CGMCC No.9286) Growth assay in copper-containing medium

[0084] Acithiobacillus thiooxidans copB Af Strains in Starkey-S 0 In liquid culture medium, shake the flask at 30°C and 180rpm to the late logarithmic period (about 5-7 days), according to the bacterial concentration of 10 6 ~10 7 A.thiooxidans copB A.f Inoculate to 100mL containing 0~100mM CuSO 4 Starkey-S 0 In liquid culture medium, shake flask culture at 30°C and 180rpm; take 1mL bacterial suspension from each bottle of bacteria for OD determination on 2 or 3 days during the culture period 600 , and plot A.thiooxidans copB Af Growth curves under different copper ion concentrations (see figure 2 ), determine A.thiooxidanscopB Af The copper ion MIC of the strain. by A. thiooxidans copB Af The growth curve of the strain under different copper ion concentra...

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Abstract

The invention discloses copper-resistant genetic engineering bacteria of acidithiobacillus ferroxidans with stable inheritance. The bacterial strain is preserved in China General Microbiological Culture Collection Center on June 9, 2014 with the preservation number of CGMCC No.9286. The engineering bacteria disclosed by the invention are obtained by integrating a copBAf gene of the acidithiobacillus ferroxidans on a genome of the acidithiobacillus ferroxidans. The invention also discloses application of the engineering bacteria in bioleaching. The experiment proves that the copper-resistant genetic engineering bacteria of the acidithiobacillus ferroxidans disclosed by the invention have higher copper resistance characteristics than wild acidithiobacillus ferroxidans, and are stable in inheritance, indicating that the copper-resistant genetic engineering bacteria have great application value in the field of bioleaching of copper ore.

Description

technical field [0001] The invention relates to a strain of acidophilic Thiobacillus thiooxidans and its application, in particular to a strain of acidophilic Thiobacillus thiooxidans capable of stably inheriting copper-resistant engineering bacteria and its application in bioleaching. Background technique [0002] Acidithiobacillus thiooxidans (A.thiooxidans) plays a very important role in the fields of bioleaching, heavy metal pollution control and biological desulfurization, but in the process of bioleaching and heavy metal pollution control, especially in copper ore During the bioleaching process, the concentration of copper ions is often very high, generally 30-90mM, and even as high as 300mM in some ore leaching environments, while the minimum inhibitory concentration (MIC) of copper ions in A.thiooxidans is usually lower than the concentration of copper ions in the above-mentioned environments For example, the copper ion MIC of A.thiooxidans ATCC19377 preserved in thi...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C22B3/18C12R1/01
CPCY02P10/20
Inventor 刘相梅文晴林建群
Owner SHANDONG UNIV
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