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Electroporation method of thermophilic thiobacillus

A thermophilic Thiobacillus, electrotransformation technology, applied in bacteria, introduction of foreign genetic material using vectors, recombinant DNA technology, etc., can solve the problems of difficult cell wall breakdown, long growth cycle, and difficult recovery.

Inactive Publication Date: 2009-02-25
SHANDONG UNIV
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  • Abstract
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Problems solved by technology

[0005] Aiming at the dense cell wall of Thiobacillus thermophile, and the viscous polysaccharides and other substances are produced around the cell wall when the cells are adsorbed and grown with sulfur powder as the energy source, the cell wall is difficult to be broken down during electroporation; the growth cycle is long, the growth of organic matter is inhibited, and the recovery is difficult, etc. , the present invention proposes a high-efficiency electrotransformation method for introducing a constructed plasmid containing an exogenous gene into Thiobacillus thermophiles

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  • Electroporation method of thermophilic thiobacillus
  • Electroporation method of thermophilic thiobacillus
  • Electroporation method of thermophilic thiobacillus

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Embodiment 1

[0022] 1. Inoculate E.coli SM10 (pJRD215) (commercially available) in Luria-Bertani medium (LB medium), use streptomycin at a concentration of 100 μg / ml, shake and culture at 200 r / min for 12 to 14 hours, collect cells, and use TaKaRaMiniBEST Plasmid Purification Kit Ver.2.0 kit (product number: DV801A) was used to extract the plasmid, and the concentration of the plasmid was accurately determined to be 112 ng / μL by an ultraviolet spectrophotometer (BioPhotometer plus) from Eppendorf Company.

[0023] 2. The thermophilic Thiobacillus MTH-04 (this laboratory was isolated from the acidic water sample of Tengchong Hot Spring, Yunnan, and the preservation number is CGMCC NO.1742; Liu Ying, Qi Fangjun, Lin Jianqun, etc. A moderately thermophilic and acidophilic strain Isolation and phylogenetic analysis of sulfur-oxidizing bacteria. Acta Microbiology, 2004, 44(3): 382-385) inoculated on Starky-S 0 In liquid medium, shake culture at 120r / min at 39°C for 5.5 days, centrifuge at 12,00...

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Abstract

The invention discloses an electro-transformation method for inducing a constructed plasmid containing exogenous genes to acidithiobacillus caldus. The method comprises the following steps: the constructed plasmid containing the exogenous genes is extracted and the concentration of the plasmid is corrected; the competent cells of the acidithiobacillus caldus are cultured and collected; the electro-transformation and plate screening are carried out, and positive transformants are identified and detected; wherein, the electro-transformation parameters are as follows: the voltage is 10,500V / cm, the resistance is 400 omega and the capacitance is 25muF. By adopting the method, the electro-transformation of the acidithiobacillus caldus is realized for the first time, and a recovery culture scheme and a screening method for the cidithiobacillus caldus are established, which provides a convenient, quick and high-efficiency gene delivery method for the studies of genetic engineering breeding, genetics and molecular biology of the acidithiobacillus caldus.

Description

technical field [0001] The invention relates to a new method for introducing exogenous genes into autotrophic microorganisms, in particular to a high-efficiency electrotransformation method for introducing constructed plasmids containing exogenous genes into thermophilic Thiobacillus. Background technique [0002] Acidithiobacillus caduls (A.caduls for short) is an acidophilic obligate autotrophic sulfur-oxidizing bacterium, and is the dominant species in biometallurgical leaching bacteria. Generally, in the well-responsive leaching solution, Thiobacillus thermophiles account for 20-30%. However, because the leaching solution often contains toxic heavy metal ions, which can inhibit it, it is necessary to use genetic engineering to construct heavy metal ion-resistant strains to meet the needs of actual production. Due to the special cell characteristics and growth environment of Thiobacillus thermophile, the gene transfer methods commonly used at present have not been succes...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N1/20
Inventor 林建群陈林旭李兵林建强
Owner SHANDONG UNIV
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