Electrotransformation of whole cells ofBrevibacteriumsp. R312 a nitrile hydratase producing strain: Construction of a cloning vector

Abstract

A rapid and effective method is described for electroporation of Brevibacterium sp. R312, a coryneform strain producing nitrile hydratase and amidase. The transformation efficiency of the method is 108 transformants per μg of plasmid under optimal conditions. Parameters optimised included field strength (11.8 kV cm−1), pulse length (2.4 ms), plasmid DNA concentration (0.25 μg ml−1 and cell density (1010 cells ml−1). Surprisingly, the transformation efficiency did not vary with the growth stage, in contrast to results in the literature. A shuttle vector was constructed containing several unique cloning sites down-stream of the SP6 RNA polymerase promoter.