Wu Shixuan, Letchworth Geoffrey J
U.S. Department of Agriculture, Laramie, WY, USA.
Biotechniques. 2004 Jan;36(1):152-4. doi: 10.2144/04361DD02.
Transformation efficiencies for Pichia pastoris are usually several orders of magnitude below those for other yeast. We report here that pretreatment of P. pastoris with 0.1 M lithium acetate (LiAc) and 10 mM dithiothreitol (DTT) before electroporation increased transformation efficiency approximately 150-fold. DTT alone enhanced the transformation efficiency up to 20-fold, but LiAc alone had little effect. Cultures grown to 1.15-2.6 A at 600 nm had higher transformation efficiencies than younger or older cultures. A cell concentration of 10(10)/mL gave the highest efficiencies. Digestion of pPIC9K within the AOX1 gene with Sacl gave efficiencies approximately 30 times higher than digestion in other genes with other enzymes. Given the optimization of these factors, the highest transformation efficiency was obtained with instrument settings of 1.5 kV, 25 microF, and 186 omega. The transformation efficiency at optimal conditions reached 4 x 10(6) transformants/microgram DNA with pPIC9K. A maximum of 2.6 x 10(5) transformants was produced when 1 microgram of pPIC9K DNA was used.
巴斯德毕赤酵母的转化效率通常比其他酵母低几个数量级。我们在此报告，在电穿孔前用0.1 M醋酸锂（LiAc）和10 mM二硫苏糖醇（DTT）对巴斯德毕赤酵母进行预处理可使转化效率提高约150倍。单独使用DTT可将转化效率提高至20倍，但单独使用LiAc效果甚微。在600 nm波长下生长至吸光度为1.15 - 2.6的培养物比年轻或年老的培养物具有更高的转化效率。细胞浓度为10¹⁰/mL时效率最高。用Sacl在AOX1基因内消化pPIC9K的效率比用其他酶在其他基因中消化高约30倍。在优化这些因素的情况下，使用1.5 kV、25 μF和186 Ω的仪器设置可获得最高转化效率。在最佳条件下，使用pPIC9K时转化效率达到4×10⁶转化子/μg DNA。当使用1 μg pPIC9K DNA时，最多可产生2.6×10⁵个转化子。
