pCVD442
(Plasmid #11074)

• Depositing Labs: Michael Donnenberg, James Kaper
• Publication: Donnenberg et al Infect Immun. 1991 Dec . 59(12):4310-7.

Backbone

• Vector backbone: pGP704
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): SY327 lambda pir
• Growth instructions: Needs pir gene (see comments below)
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: sacB
• Species: Bacillus subtilis
• Insert Size (bp): 1400
• Entrez Gene: sacB (a.k.a. BSU_34450, BSU34450)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: PstI (not destroyed)
• 3′ cloning site: PstI (not destroyed)
• 5′ sequencing primer: na

Resource Information

• Addgene Notes:
  • Addgene Diagnostic Digest
• Articles Citing this Plasmid:
  • 6 References

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor 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.