Novel plasmid transformation method mediated by chrysotile, sliding friction, and elastic body exposure.
Escherichia coli as a plasmid recipient cell was dispersed in a chrysotile colloidal solution, containing chrysotile adsorbed to plasmid DNA (chrysotile-plasmid cell mixture). Following this, the chrysotile-plasmid cell mixture was dropped onto the surface of an elastic body, such as agarose, and treated physically by sliding a polystyrene streak bar over the elastic body to create friction. Plasmid DNA was easily incorporated into E. coli, and antibiotic resistance was conferred by transformation. The transformation efficiency of E. coli cultured in solid medium was greater than that of E. coli cultured in broth. To obtain greater transformation efficiency, we attempted to determine optimal transformation conditions. The following conditions resulted in the greatest transformation efficiency: the recipient cell concentration within the chrysotile-plasmid cell mixture had an optical density greater than or equal to 2 at 550 nm, the vertical reaction force applied to the streak bar was greater than or equal to 40 g, and the rotation speed of the elastic body was greater than or equal to 34 rpm. Under these conditions, we observed a transformation efficiency of 10(7) per microg plasmid DNA. The advantage of achieving bacterial transformation using the elastic body exposure method is that competent cell preparation of the recipient cell is not required. In addition to E. coli, other Gram negative bacteria are able to acquire plasmid DNA using the elastic body exposure method.
Yoshida
Naoto
N
Department of Biochemistry and Applied Biosciences, University of Miyazaki, Miyazaki-shi, 889-2192, Japan. a04109u@cc.miyazaki-u.ac.jp
Nakajima-Kambe
Toshiaki
T
Matsuki
Kaori
K
Shigeno
Toshiya
T