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pCVD442
(Plasmid #11074)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 11074 Standard format: Plasmid sent in bacteria as agar stab 1 $75

This material is available to academics and nonprofits only.

Backbone

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
  • (Common Sequencing Primers)

Resource Information

Terms and Licenses

  • Academic/Nonprofit Terms
  • Industry Terms
    • Not Available to Industry
Trademarks:
  • Zeocin® is an InvivoGen trademark.

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.

How to cite this plasmid ( Back to top)

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.

  • For your Materials & Methods section:

    pCVD442 was a gift from Michael Donnenberg & James Kaper (Addgene plasmid # 11074 ; http://n2t.net/addgene:11074 ; RRID:Addgene_11074)
  • For your References section:

    Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Donnenberg MS, Kaper JB. Infect Immun. 1991 Dec . 59(12):4310-7. PubMed 1937792