An improved vector system for insertional gene inactivation inspired by the tmRNA-tagging system of S. pneumoniae
| Autor: | Juliette Molnos, Kurt Amrein, Roland Lange |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Microbiology (medical)
Genetics Recombination Genetic Genetic Vectors Translation (biology) Biology Microbiology Insertional mutagenesis chemistry.chemical_compound Mutagenesis Insertional RNA Bacterial Plasmid Streptococcus pneumoniae chemistry Bacterial Proteins Gene Targeting Vector (molecular biology) Insertion Site-directed mutagenesis Molecular Biology Gene DNA Gene Deletion Plasmids |
| Zdroj: | Journal of microbiological methods. 42(2) |
| ISSN: | 0167-7012 |
| Popis: | Insertional mutagenesis is a technique often used to inactivate genes in Streptococcus pneumoniae. Using conventional vectors, a 5' segment of the targeted gene remains under the control of the gene's authentic promoter following gene disruption. Thus, the expression of a functional peptide and the misinterpretation of results in consequence cannot be excluded. To circumvent this problem, we have developed a plasmid for insertional mutagenesis based on the tmRNA-tagging system of S. pneumoniae which ensures that any protein expressed after gene disruption is degraded. Insertional mutagenesis using this vector results in the targeted gene being tagged with a tmRNA-derived sequence coding for a proteolysis tag. Here we show that the translation product of a gene tagged by this method is not detectable by Western blotting, suggesting that the protein was degraded. This modified vector allows total inactivation of genes with a reliability that cannot be achieved by conventional vectors for insertional mutagenesis. This approach can be applied to other bacterial species. |
| Databáze: | OpenAIRE |
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