URL: http://www.addgene.org/11074

Proper Citation: RRID:Addgene_11074

Insert Name: sacB

Organism: Bacillus subtilis

Bacterial Resistance: Ampicillin

Defining Citation: PMID:1937792

Vector Backbone Description: Backbone Size:0; Vector Backbone:pGP704; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin

Comments: This suicide vector was created to engineer mutations in host strains via allelic exchange. The important properties of this vector are: 1) a pir dependent origin of replication from plasmid R6K, 2) the bla gene encoding resistance to ampicillin, 3) the mob region allowing efficient transfer by conjugation from strains containing the tra locus, and 4) the sacB gene conferring sensitivity to sucrose in Gram-negative bacteria. Plasmid pCVD442 was created by cloning the sacB gene on a PstI fragment from plasmid pUM24 (Ried and Collmer. Gene 57:239-46, 1987) into plasmid pGP704 (Miller and Mekalanos. J.Bacteriol. 170:2575-83, 1988) that had been partially digested with PstI. Plasmid pCVD442 and its derivatives can only grow in strains that have the pir gene encoding the Pi protein, which is necessary for replication of R6K plasmids. The pir gene is usually supplied by a lambda lysogen. Such strains include DH5alpha-lambdapir, SY327-lambdapir, SM10-lambdapir, and S17-lambdapir. The last two strains supply the tra genes for efficient conjugation. Sucrose sensitivity is far from absolute. The strain carrying the plasmid grows well on sucrose plates. To detect sucrose sensitivity make serial dilutions of the bacteria and plate both on plates containing and lacking 5% sucrose. There should be substantially fewer colonies and the colonies are smaller on the sucrose plates.

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