Cutrín J M, Conchas R F, Barja J L, Toranzo A E
Departamento de Microbiología y Parasitología, Facultad de Biología, Universidad de Santiago de Compostela.
Microbiologia. 1994 Mar-Jun;10(1-2):69-82.
Yersinia ruckeri, a fish pathogenic bacterium in aquaculture, was used to evaluate the electroporation as a new transformation method for this species. DNA used for the electrotransformation were plasmids of molecular mass ranging from 2.3 kb to 33 kb, and diverse replicons. To optimize this method we used Y. ruckeri 11.29 strain (from serotype 02) and pSU2718 DNA. The best transformation efficiency (6.0 x 10(5) transformants/micrograms DNA) was obtained with 12.5 kV/cm, 25 microF, 400 omega and 2 hours of incubation after pulse. When these conditions were applied to other strains belonging to different serotypes and other plasmids, we obtained transformants in all strains assayed, but only when using low molecular weight plasmids. Plasmid vectors and resident plasmid were not modified in host strains after electrotransformation. In studies of conformation we confirmed that only circular DNA was able for transformation. The utilization of this technique for direct cloning in Y. ruckeri makes possible further studies on recombinant DNA.
鲁氏耶尔森菌是水产养殖中的一种鱼类致病细菌，被用于评估电穿孔作为该菌种新的转化方法。用于电转化的DNA是分子量范围从2.3 kb到33 kb的质粒以及不同的复制子。为优化此方法，我们使用了鲁氏耶尔森菌11.29菌株（血清型02）和pSU2718 DNA。在12.5 kV/cm、25 μF、400 Ω的条件下以及脉冲后孵育2小时，获得了最佳转化效率（6.0×10⁵转化子/微克DNA）。当将这些条件应用于属于不同血清型的其他菌株和其他质粒时，我们在所检测的所有菌株中都获得了转化子，但仅在使用低分子量质粒时。电转化后宿主菌株中的质粒载体和常驻质粒未被修饰。在构象研究中，我们证实只有环状DNA能够进行转化。这种技术在鲁氏耶尔森菌中用于直接克隆使得对重组DNA的进一步研究成为可能。
