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Thermophilic thiobacillus gene engineering bacterium with mercury resistant characteristic and application thereof

A technology of thermophilic Thiobacillus and genetically engineered bacteria, which is applied in the direction of bacteria and process efficiency improvement, can solve the problems of slow growth, inconvenient research work, limited application range, etc., and achieve the effect of improving mercury resistance

Inactive Publication Date: 2007-08-15
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the actual application process, the slow growth of the bacteria, the low cell yield and the lack of resistance to heavy metals such as arsenic, mercury, silver and other weaknesses limit its application range and bring a lot of inconvenience to the research work of the laboratory.
According to domestic and foreign literature search, there is no report about genetically modifying the bacteria and successfully constructing mercury-resistant genetically engineered bacteria.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Construction and identification of mercury-resistant plasmid pTMJ212.

[0033] (1) After linking the mercury-resistant fragment from pTM314 to the shuttle plasmid pJRD215, transform Escherichia coli SM10 in the presence of Sm / HgCl 2 Escherichia coli SM10 containing the Sm-resistant plasmid was screened on the LB solid plate, and it was verified by digestion of the extracted plasmid that the plasmid carried by the transformant did contain the mercury-resistant fragment merCA.

[0034] (2) After studying the anti-mercury properties of Escherichia coli SM10 (pTMJ212), it was shown that the anti-mercury properties of merCA were successfully expressed in the recombinant plasmid pTMJ212, making E. coli SM10 resistant to HgCl 2 The resistance of the drug was increased from 10μg / ml to 22.5μg / ml.

[0035] In the above LB screening medium, Sm, HgCl 2 The screening concentrations were 100 μg / ml and 18 μg / ml, respectively.

Embodiment 2

[0036] Example 2: Construction and identification of Thiobacillus thermophilicus mercury-resistant genetically engineered bacteria.

[0037] (1) Collect Escherichia coli SM10 (pTMJ212) in the mid-exponential phase as the donor bacteria, wild-type Thiobacillus thermophilic MTH-04 in the stable phase as the recipient bacteria, wash 3 times with inorganic salt washing solution, and suspend at the same bacterial concentration Use in washing liquid for later use.

[0038] (2) According to the volume ratio of the donor bacteria and the recipient bacteria as 2:1, mix the suspension, take 150ul and put it on a nitrocellulose filter membrane with a pore size of 0.45μm, and cultivate it on a joint plate at 37°C for 48h; Wash the bacteria on the filter membrane with 1ml of washing solution and dilute to 10 -3 、10 -4 , respectively take 200μl coating Starky-Na 2 S 2 o 3 Screen the plate and culture it statically at 42°C for 10 days.

[0039] (3) Pick the resistant colonies on the sc...

Embodiment 3

[0042] Example 3: Species identification and preservation information of Thiobacillus thermophilic mercury-resistant genetically engineered bacteria.

[0043] The mercury-resistant Thiobacillus thermophilic strain obtained in the manner of Example 2 was preserved on June 26, 2006 in the General Microbiology Center of China Microbiological Culture Collection Management Committee (Institute of Microbiology, Chinese Academy of Sciences, Beijing, China), and the preservation center number is: CGMCC1742 .

[0044] The above-mentioned Thiobacillus thermophile CGMCC1742, after identification, has the following biological characteristics:

[0045] It has thermophilic and acidophilic characteristics, Gram-negative, terminal flagella, no movement, strictly aerobic, obligate autotrophic sulfur-oxidizing bacteria, short rod-shaped, with a size of (0.6-0.8) μm×(1 ~2) μm; using sulfur or sulfur complexes as energy sources, it can grow on solid medium containing sodium thiosulfate and potas...

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PUM

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Abstract

The invention discloses a preparing method of strain thermophilic thiobacilleae with anti-mercury property with keeping number as CGMCC 1742, which comprises the following steps: (1) constructing anti-mercury plasmid carrier; (2) constructing and attesting anti-mercury genetic engineering bacterium; (3) culturing the seed of A.caldus CGMCC 1742; (4) enlarging culture A.caldus CGMCC 1742 (5) growing in mercury culture medium; testing the anti-mercury property. This product possesses stable anti-mercury property, which can be used widely in biological dipping domain.

Description

technical field [0001] The invention relates to a strain of thermophilic thiobacterium and its application, in particular to a strain of thermophilic thiobacterium with mercury-resistant properties and its application. Background technique [0002] Acithiobacillus caldus plays a very important role in bacterial leaching. In the actual application process, the slow growth of the bacteria, low cell yield and lack of resistance to heavy metals such as arsenic, mercury, silver and other weaknesses limit its application range and bring a lot of inconvenience to the laboratory research work. Searching domestic and foreign literature, there is no report about the successful construction of mercury-resistant genetically engineered bacteria by genetically modifying the bacteria. Contents of the invention [0003] In view of the lack of resistance of the bacteria to mercury in the prior art, the problem to be solved in the present invention is to provide a strain of Thiobacillus th...

Claims

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Application Information

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IPC IPC(8): C12N1/20C22B3/18
CPCY02P10/20
Inventor 林建群陈丹丹林建强刘相梅颜望明
Owner SHANDONG UNIV
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