pLR16-Phes*
(Plasmid #98783)

• Purpose: Suicide vector for allelic exchange in Listeria utilizing an engineered counterselection marker for Listeria monocytogenes based on a mutated phenylalanyl-tRNA synthetase gene (pheS*).
• Depositing Lab: Anat Herskovits
• Publication: Argov et al Appl Environ Microbiol. 2017 Mar 2;83(6). pii: e02927-16. doi: 10.1128/AEM.02927-16. Print 2017 Mar 15.

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:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

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.