Electroporation method of thermophilic thiobacillus

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 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 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 successful: use CaCl ...

AI Technical Summary

Problems solved by technology

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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