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URL: http://www.addgene.org/98783

Proper Citation: RRID:Addgene_98783

Insert Name: pheS

Organism: Bacillus subtillis

Bacterial Resistance: Chloramphenicol

Defining Citation: PMID:28039138

Vector Backbone Description: Vector Backbone:An pKSV7oriT derivative vector (pBHE261) and pPL2; Vector Types:Bacterial Expression; Bacterial Resistance:Chloramphenicol

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.

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