有机溶剂对野生型枯草芽胞杆菌转化效率的影响

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Abstract Bacillus subtilis is a probiotics that can be added directly to the diet to improve the uptake of nutrients in animals. With the rapid development of gene technology, the expression system of B. subtilis is widely used in the livestock industry. In order to ameliorate the transformation efficiency of B. subtilis competent cells, different types and concentrations organic solvents were added in the process of competent cells preparation to optimize the transformation efficiency. In this study, 1.0%, 2.0%, 3.0%, 4.0% and 5.0% of the Tween-80, methanol and acetone were added in preparation of wild-type Bacillus subtilis LN competent cell. The number of viable bacteria was calculated by dilution plate counting method to determine the best addition reagent and its concentration. The plasmid pGEM-kpgt and pGEM-kmpgt with the length of 500 and 2 680 bp was transformed into individual competent cells. The transformation results were determined by PCR using P4326F/P4326R and KgF/KgR as primers and the transformation efficiency was calculated. The results showed that the count of viable competent cell is 546±13 cell/μL and the highest transformation efficiency is 52±4 transformants/μg DNA when adding 4% methanol in the preparation process. When the length of the target gene fragment was extended to 2~3 kb, the transformation efficiency was 29±2 transformants/μg. Through the optimization of the preparation method of the competent cell, the transformation efficiency of the competent cell of Bacillus subtilis was improved, which provides a potential tool for genetic engineering of Bacillus subtilis.

Key words： wild-type B. subtilis organic solvent competent cells number of resurrection cells transformation efficiency

Received: 20 March 2017 Published: 01 November 2017

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	Articles by authors
	ZHE Li-Bei
	LI Wang
	SHI Tuan-Qing
	SONG Xiang-Xiang
	YU Tie-Ban

Cite this article:

ZHE Li-Bei,LI Wang,SHI Tuan-Qing, et al. Effect of Organic Solvent on the Transformation Efficiency of Wild-type Bacillus Subtilis[J]. , 2017, 25(11): 1878-1886.

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http://journal05.magtech.org.cn/Jwk_ny/EN/ OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2017/V25/I11/1878

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