A comprehensive toolbox for the rapid construction of lacZ fusion reporters
| Autor: | Jürgen Lassak, Kirsten Jung, Luitpold Fried |
|---|---|
| Rok vydání: | 2012 |
| Předmět: |
Recombination
Genetic Microbiology (medical) Ribosomal Protein S9 Escherichia coli Proteins Recombinant Fusion Proteins Genetic Vectors lac operon Biology beta-Galactosidase medicine.disease_cause Microbiology Molecular biology Cell biology White (mutation) Lac Operon Genes Reporter Gene expression Escherichia coli Recombinase medicine Cloning Molecular Lacz reporter Homologous recombination Molecular Biology |
| Zdroj: | Journal of Microbiological Methods. 91:537-543 |
| ISSN: | 0167-7012 |
| Popis: | β-Galactosidase encoded by lacZ remains a popular reporter enzyme. Here, we present three fast and convenient tools that facilitate rapid construction of reporter lacZ fusions. The first enables the simple generation of lacZ (slacZ)-based chromosomally encoded reporter fusions within the lac operon in Escherichia coli using Red®/ET® recombination. The slacZ tool is based on rpsL counter-selection in combination with homologous recombination catalyzed by the λ Red recombinase, and blue/white screening. This permits construction of transcriptional and translational reporter lacZ fusions within a day. The second tool allows the introduction of lacZ reporter fusions into the chromosome by a single-crossover method. The strategy relies on the γ-origin-based suicide vector pNPTS138-R6KT, which can only replicate in λpir E. coli strains. The third tool comprises four pBBR1-based broad-host-range vectors for transcriptional and translational lacZ fusions. The functionality of our toolbox was confirmed by the K(+)-dependent activation of kdp promoter-lacZ fusions in vivo. |
| Databáze: | OpenAIRE |
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