Plasmid pIJ699, a multi-copy positive-selection vector for Streptomyces

Abstract

A plasmid vector, pIJ699, which provides positive selection for cloned DNA, was constructed using the replication functions of the Streptomyces wide-host-range multi-copy plasmid pIJ101. The selection for inserts is based on the principle that plasmids with long uninterrupted perfect palindromes (inverted repeats) are 'not viable' in bacteria. For cloning, pIJ699 is digested with BglII. This produces two fragments, one of which is the linearized vector, with two arms of the palindrome at its ends, and the other is a 'spacer' which is needed to keep the inverted repeat sequences apart. The vector fragment is separated from the 'spacer' fragment and ligated with the DNA to be cloned. Plasmids with a fragment of cloned DNA, but not the circularized vector, give rise to thiostrepton-resistant transformants in Streptomyces lividans. The inverted repeat sequences contain a strong transcription terminator which reduces transcriptional read-through both in and out of the cloned fragment. This improves the stability of many hybrid plasmids and facilitates the study of the regulation of cloned genes.