Exploration of Pichia pastoris Electroporation Method

Abstract

Abstract: This paper aims to improve the transformation efficiency of Pichia pastoris by optimized electroporation. The transformation was carried on with different pretreatment buffer, cell volume, DNA amounts and electroporation voltage. The transformation efficiency was defined by counting the numbers after cultivating and calculating the clones per μg DNA. Research showed that a better transformation efficiency can be obtained when the pretreatment buffer is 0.1 mol/L LiAc, 0.01 mol/L DTT, 0.6 mol/L sorbitol, 0.01 mol/L Tris-HCl and the DNA amount is 10 ng. For the 0.1 cm cuvette, the highest transformation efficiency was 9.6×10⁵ /μg DNA when the electroporation voltage was 0.6 kV and the cell volume was 80 μL. For the 0.2 cm cuvette, the highest transformation efficiency was 8.8×10⁵/μg DNA when the electroporation voltage was 1.1 kV and the cell volume was 150 μL. At the same time, we compared the transformation efficiency of different type of plasmid and found that the transformation efficiency of pPIC9K was 160 times than that of pPICZαA and 2 900 times than that of pGAPZαA. By applying this developed protocol, it have improved the transformation efficiency of Pichia pastoris from 10^3-4 to 10⁵ / μg DNA.

Key words: Pichia pastoris, Electroporation, Transformation efficiency

Yang Xiaopan, Dong Lihou, Wan Deyou, Hua Ling, Liu Yunhui, Gao Xin. Exploration of Pichia pastoris Electroporation Method[J]. Biotechnology Bulletin, 2013, 0(12): 156-161.

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