Development of a broad-host-range sacB-based vector for unmarked allelic exchange

The development of an improved, broad-host-range "in-out" allelic exchange vector, pCM433, which permits the generation of clean, marker-free genetic manipulations, and has the advantages of a high copy, general Escherichia coli replicon for easy cloning and an IncP oriT enabling conjugal transfer.

Abstract

The broad-host-range vector for marker-free allelic exchange described here, pCM433, has the advantages of a high copy, general Escherichia coli replicon for easy cloning, an IncP oriT enabling conjugal transfer, an extensive set of restriction sites in its polylinker, three antibiotic markers, and sacB (encoding levansucrase) for negative selection upon sucrose plates. These traits should permit pCM433 to be broadly applied across many bacterial taxa for marker-free allelic exchange, which is particularly important if multiple manipulations or more subtle genetic manipulations such as point mutations are desired.