ELECTROTRANSFORMATION OF WHOLE CELLS OF BREVIBACTERIUM SP R312 A NITRILE HYDRATASE PRODUCING STRAIN - CONSTRUCTION OF A CLONING VECTOR

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 10(8) transformants per-mu-g of plasmid under optimal conditions. Parameters optimised included field strength (11.8 kV cm-1), pulse length (2.4 ms), plasmid DNA concentration 90.25-mu-g ml-1) and cell density (10(10) 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 downstream of the SP6 RNA polymerase promoter.