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Addgene

pLR16-Phes*
(Plasmid #98783)

Ordering

This material is available to academics and nonprofits only.
Item Catalog # Description Quantity Price (USD)
Plasmid 98783 Standard format: Plasmid sent in bacteria as agar stab 1 $85

Backbone

  • Vector backbone
    An pKSV7oriT derivative vector (pBHE261) and pPL2
  • Vector type
    Bacterial Expression

Growth in Bacteria

  • Bacterial Resistance(s)
    Chloramphenicol, 25 μg/mL
  • Growth Temperature
    37°C
  • Growth Strain(s)
    DH5alpha
  • Copy number
    High Copy

Gene/Insert

  • Gene/Insert name
    pheS
  • Species
    Bacillus subtillis
  • Mutation
    A309G

Terms and Licenses

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

Depositor Comments

Construction of Listeria monocytogenes mutants by allelic exchange has been laborious and time-consuming due to lack of proficient selection markers for the final recombination event, that is, a marker conveying substance sensitivity to the bacteria bearing it, enabling the exclusion of merodiploids and selection for plasmid loss. In order to address this issue, we engineered a counterselection marker based on a mutated phenylalanyl-tRNA synthetase gene (pheS*). This mutation renders the phenylalanine-binding site of the enzyme more promiscuous and allows the binding of the toxic p-chloro-phenylalanine analog (p-Cl-phe) as a substrate. When pheS* is introduced into L. monocytogenes and highly expressed under control of a constitutively active promoter, the bacteria become sensitive to p-Cl-phe supplemented in the medium. This enabled us to utilize pheS* as a negative selection marker and generate a novel, efficient suicide vector for allelic exchange in L. monocytogenes.

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:

    pLR16-Phes* was a gift from Anat Herskovits (Addgene plasmid # 98783 ; http://n2t.net/addgene:98783 ; RRID:Addgene_98783)
  • For your References section:

    An Effective Counterselection System for Listeria monocytogenes and Its Use To Characterize the Monocin Genomic Region of Strain 10403S. Argov T, Rabinovich L, Sigal N, Herskovits AA. Appl Environ Microbiol. 2017 Mar 2;83(6). pii: e02927-16. doi: 10.1128/AEM.02927-16. Print 2017 Mar 15. 10.1128/AEM.02927-16 PubMed 28039138