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PurposeSuicide vector for allelic exchange in Listeria utilizing an engineered counterselection marker for Listeria monocytogenes based on a mutated phenylalanyl-tRNA synthetase gene (pheS*).
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Depositing Lab
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Sequence Information
Ordering
Item | Catalog # | Description | Quantity | Price (USD) | |
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Plasmid | 98783 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $85 |
Backbone
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Vector backboneAn pKSV7oriT derivative vector (pBHE261) and pPL2
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Vector typeBacterial Expression
Growth in Bacteria
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Bacterial Resistance(s)Chloramphenicol, 25 μg/mL
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Growth Temperature37°C
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Growth Strain(s)DH5alpha
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Copy numberHigh Copy
Gene/Insert
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Gene/Insert namepheS
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SpeciesBacillus subtillis
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MutationA309G
Terms and Licenses
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Academic/Nonprofit Terms
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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.
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.
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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