Abstract
BackgroundAlthough genome sequences are available for an ever-increasing number of bacterial species, the availability of facile genetic tools for physiological analysis have generally lagged substantially behind traditional genetic models.ResultsHere I describe the development of an improved, broad-host-range "in-out" allelic exchange vector, pCM433, which permits the generation of clean, marker-free genetic manipulations. Wild-type and mutant alleles were reciprocally exchanged at three loci in Methylobacterium extorquens AM1 in order to demonstrate the utility of pCM433.ConclusionThe 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.
Highlights
Genome sequences are available for an ever-increasing number of bacterial species, the availability of facile genetic tools for physiological analysis have generally lagged substantially behind traditional genetic models
This paper presents a facile, broad-host-range "in-out" system based on sacB that has been designed to allow facile unmarked allelic exchange in a wide variety of bacterial taxa
Construction of the "in-out" allelic exchange vector pCM433 In order to generate a facile system for marker-free allelic exchange across a wide variety of bacterial species, the loxP-flanked kanamycin (Km) resistance cassette of the broad-host-range marker-recycling vector, pCM184 [4] was first excised and replaced with a synthetic linker that introduced three new restriction sites to the extensive multiple-cloning sites
Summary
I describe the development of an improved, broad-host-range "in-out" allelic exchange vector, pCM433, which permits the generation of clean, marker-free genetic manipulations. Wild-type and mutant alleles were reciprocally exchanged at three loci in Methylobacterium extorquens AM1 in order to demonstrate the utility of pCM433
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