Liebl W, Bayerl A, Schein B, Stillner U, Schleifer K H
Lehrstuhl für Mikrobiologie, Technische Universität München, F.R.G.
FEMS Microbiol Lett. 1989 Dec;53(3):299-303. doi: 10.1016/0378-1097(89)90234-6.
High-frequency electroporation of whole Corynebacterium glutamicum cells without enzymatic pretreatment was achieved. Under optimized conditions concerning growth stage, washing of cells, cell concentration and pulse parameter transformation efficiencies of far more than 10(7) transformants per microgram pWST4B plasmid DNA were reached. Using electroporation, linearised and subsequently religated plasmid as well as chimeric ligase reaction products were directly introduced into C. glutamicum with reasonable efficiencies. Electrotransformation efficiency was reduced about 10(5)-fold for plasmid DNA cycled through E. coli JM83. Restriction deficient mutants of C. glutamicum were isolated which could be efficiently transformed with foreign DNA.
实现了对谷氨酸棒杆菌全细胞进行无酶预处理的高频电穿孔。在关于生长阶段、细胞洗涤、细胞浓度和脉冲参数的优化条件下，每微克pWST4B质粒DNA的转化效率远远超过10⁷个转化体。使用电穿孔，线性化并随后重新连接的质粒以及嵌合连接酶反应产物以合理的效率直接导入谷氨酸棒杆菌。对于通过大肠杆菌JM83循环的质粒DNA，电转化效率降低了约10⁵倍。分离出了谷氨酸棒杆菌的限制缺陷型突变体，其能够被外源DNA高效转化。
