van der Rest M E, Lange C, Molenaar D
Heinrich-Heine-Universität, Düsseldorf, Germany.
Appl Microbiol Biotechnol. 1999 Oct;52(4):541-5. doi: 10.1007/s002530051557.
An improved method for the electrotransformation of wild-type Corynebacterium glutamicum (ATCC 13032) is described. The two crucial alterations to previously developed methods are: cultivation of cells used for electrotransformation at 18 degrees C instead of 30 degrees C, and application of a heat shock immediately following electrotransformation. Cells cultivated at sub optimal temperature have a 100-fold improved transformation efficiency (10(8) cfu micrograms-1) for syngeneic DNA (DNA isolated from the same species). A heat shock applied to these cells following electroporation improved the transformation efficiency for xenogeneic DNA (DNA isolated from a different species). In combination, low cultivation temperature and heat shock act synergistically and increased the transformation efficiency by four orders of magnitude to 2.5 x 10(6) cfu micrograms-1 xenogeneic DNA. The method was used to generate gene disruptions in C. glutamicum.
描述了一种改进的野生型谷氨酸棒杆菌（ATCC 13032）电转化方法。与先前开发的方法相比，有两个关键改变：用于电转化的细胞在18℃而非30℃下培养，以及在电转化后立即进行热激处理。在亚最佳温度下培养的细胞对于同基因DNA（从同一物种分离的DNA），其转化效率提高了100倍（10⁸ cfu μg⁻¹）。对这些细胞进行电穿孔后施加热激处理，提高了异源DNA（从不同物种分离的DNA）的转化效率。低温培养和热激处理协同作用，将异源DNA的转化效率提高了四个数量级，达到2.5×10⁶ cfu μg⁻¹。该方法用于在谷氨酸棒杆菌中产生基因破坏。
