An improved suicide vector for construction of chromosomal insertion mutations in bacteria

We have constructed an R6K-based suicide vector (pJP5603) that requires a trans supply of the pir-encoded pi protein of plasmid R6K for replication. Therefore, efficient plasmid suicide results upon transfer to bacteria not harbouring pir. The 3.1-kb vector encodes kanamycin resistance and is mobilizable. When used in conjunction with a JM109 strain carrying pir, it has nine unique restriction sites available for alpha-complementation cloning. Vector functionality was demonstrated in Rhodobacter sphaeroides.