Donnenberg Lab Resources

Positive Selection Suicide Vector pCVD442

Description
Procedures
Requests

Description

     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
  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.  The plasmid has not been sequenced, but a restriction map appears below.  Unique sites include SalI, SphI, SacI, and SmaI (blunt).  XbaI has two sites, but can be used with the enzymes that have unique sites except for SalI.

    Plasmid pCVD442 has been requested by over 50 laboratories, has been referenced in more than 100 publications, and has been successfully used in numerous organisms including E. coli, Salmonella typhimurium, S. typhi, Shigella flexneri, Proteus mirabilis, Vibrio cholerae, V. vulnificus and even (via E. coli) Mycobacterium tuberculosis.

Procedures

Propagation of the plasmid

        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.

Cloning into pCVD442

    Cloning in pCVD442 may not be as effecient as cloning in other vectors.  Plasmid pCVD442 has only a few unique sites for cloning (see above).  Additionally, transformation of pir containing strains might not be as efficient as is transformation of other strains.  When we have difficulty cloning into this plasmid, we sometimes create a hybrid plasmid by joining the plasmid containing the allele we wish to clone to the suicide vector with pCVD442.  If the plasmid containing the desired allele does not encode ampicillin resistance, one can transform a high efficiency strain and select for ampicillin resistance.  Since pCVD442 is unable to grow in a strain that lacks pir, the only ampicillin resistant strains that arise contain the hybrid plasmid.  After confirming the orientation, we excise the origin of replication of the original vector and transform DH5alpha-lambdapir.  Since this is an intramolecular ligation, it is highly efficient.

    It is important to have sufficient flanking DNA around the altered allele to allow recombination on both sides.  It is ideal to have greater than 1 kb on either side.  Having equal amounts of DNA on each side is also important.  Recombination is more likely to occur on the side with longer flanking DNA.  Since allelic exchange requires recombination first on one side and then on the other, it is important to have equal lengths on each side.  One can get away with less flanking DNA and with unequal amounts when one is using an allele that has a selectable marker.

Tranferring the suicide vector into the host strain; selecting for the first recombination event

    Plasmid pCVD442 containing the allele of inerest can be introduced into the parental strain by a variety of methods including electroporation, direct conjugation from a strain that carries tra functions, or triparental conjugation along with a strain that carries a broad host-range RP4 type plasmid.

Selection for loss of the suicide vector

    Once a partial merodipoid strain containing the integrated suicide vector has been obtained, the next step is a second recombination event that results in loss of the suicide vector and one of the alleles.  To select for this event we dilute an overnight LB + ampcillin culture 10-6 in LB without ampicillin and grow until the culture becomes turbid.  Serial dilutions are then plated on LB plates and on plates that contain modified LB that has no NaCl and has 5% sucrose.  These plates are incubated overnight at 30oC.  Suprisingly, the difference in the number of colonies on the plates containing and lacking sucrose is usually only ~10-fold, but most of the sucrose-resistant colonies are also ampicillin sensitive, indicating loss of the suicide vector.  These colonies can then be checked by Southern blot or PCR to determine whether they have retained the wild type or the mutated allele.

Problems?
    Although we have never encountered this problem, it has been reported that insertion of the plasmid into an unintended site can occur due to a partial insertion element present in pCVD442 (ref).



To request this plasmid please contact the folks at Addgene.  For other plasmids or strains, please contact Xiaolin Wang.


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