Study on the Electrotransformation Conditions of Lactobacillus delbrueckii subsp. bulgaricus Using Protoplasts

被引：0

作者：

Guo X. ^{[1
]}

Wang S. ^{[1
]}

Li C. ^{[1
]}

Lu H. ^{[1
]}

Tian H. ^{[1
]}

Luo Y. ^{[2
]}

机构：

[1] College of Food Science and Technology, Agricultural University of Hebei, Baoding, 071001, Hebei

[2] College of Food Science and Nutritional Engineering, China Agricultural University, Beijing

来源：

Journal of Chinese Institute of Food Science and Technology | 2019年 / 19卷 / 03期

关键词：

Electrotransformation; Lactobacillus delbrueckii subsp. bulgaricus; Plasmid pMG36e; Transformational efficiency;

D O I：

10.16429/j.1009-7848.2019.03.021

中图分类号：

学科分类号：

摘要：

Objective: The effects of different factors on the electroporation efficiency of Lactobacillus bulgaricus protoplasts were studied. The optimum conditions of electroporation were studied to improve the efficiency of electrotransformation. Methods: Lactobacillus delbrueckii subsp. bulgaricus was used as the host, and the E. coli - lactic acid bacteria shuttle plasmid pMG36e were used as the vector. The effects of the concentration of lysozyme, the concentration of plasmid, the parameters of the transformation and the composition of the recovery medium on the efficiency were studied by the method of protoplast transformation. Results: The cells were hydrolyzed by SMM at 37℃ with lysozyme(30 mg/mL) until approximately 60% of the protoplasts were found by microscopy. 1 μL of plasmid pMG36e with a concentration of 1.2 μg/μL was used for electric shock at the parameters of 8 kV/cm, 200 Ω and 25 μFu U. The cells were incubated in MRS medium containing 0.5 mol/L sucrose and 20 mmol/L MgCl2, CaCl2 (without Tween 80) for 2.5 hours, and screened in MRS medium containing erythromycin, the best electroporation efficiency was obtained. As a result, the transformational efficiency reaches 1.66×105 cfu/μg DNA. Conclusion: This study achieved efficient genetic transformation of Lactobacillus bulgaricus and provided technical support for genetic breeding. © 2019, Editorial Office of Journal of CIFST. All right reserved.

引用

页码：160 / 166

页数：6

共 5 条

[1]

Patnaik R., Louie S., Gavrilovic V., Et al., Genome shuffling of Lactobacillus for improved acid tolerance, Nature Biotechnology, 20, 7, pp. 707-712, (2002)

[2]

Neumann E., Rosenheck K., Permeability changes induced by electric impulses in vesicular membranes, The Journal of Membrane Biology, 10, 1, pp. 279-290, (1972)

[3]

McIntyre D.A., Harlander S.K., Improved electroporation efficiency of intact Lactococcuslactis subsp. Lactis Cells Grown in Defined media, Applied and Environmental Microbiology, 55, 10, pp. 2621-2626, (1989)

[4]

Serror P., Sasaki T., Ehrlich S.D., Et al., Electrotransformation of Lactobacillus delbrueckii subsp. Bulgaricus and L. Delbrueckii Subsp. Lactis with Various plasmids, Applied and Environmental Microbiology, 68, 1, pp. 46-52, (2002)

[5]

Wei M.Q., Rush C.M., Norman J.M., Et al., An improved method for the transformation of Lactobacillus strains using electroporation, Journal of Microbiological Methods, 21, 3, pp. 97-109, (1995)

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