Improving the stability of the TetR/Pip-OFF mycobacterial repressible promoter system
| Autor: | Saber Anoosheh, Laura Cioetto Mazzabò, Francesca Boldrin, Agnese Serafini, Roberta Provvedi, Riccardo Manganelli, Giorgio Palù |
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| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Genetic Vectors Mutant lcsh:Medicine Repressor Computational biology Biology Article Frameshift mutation 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Plasmid Bacterial Proteins Gene expression TetR Cloning Molecular lcsh:Science Promoter Regions Genetic Gene Regulation of gene expression Multidisciplinary Integrases lcsh:R Gene Expression Regulation Bacterial Mycobacterium tuberculosis 3. Good health Repressor Proteins 030104 developmental biology chemistry Tetracyclines lcsh:Q 030217 neurology & neurosurgery |
| Zdroj: | Scientific Reports Scientific Reports, Vol 9, Iss 1, Pp 1-6 (2019) |
| ISSN: | 2045-2322 |
| Popis: | Tightly regulated gene expression systems are powerful tools to study essential genes and characterize potential drug targets. In a past work we reported the construction of a very stringent and versatile repressible promoter system for Mycobacterium tuberculosis based on two different repressors (TetR/Pip-OFF system). This system, causing the repression of the target gene in response to anhydrotetracycline (ATc), has been successfully used in several laboratories to characterize essential genes in different mycobacterial species both in vitro and in vivo. One of the limits of this system was its instability, leading to the selection of mutants in which the expression of the target gene was no longer repressible. In this paper we demonstrated that the instability was mainly due either to the loss of the integrative plasmid carrying the genes encoding the two repressors, or to the selection of a frameshift mutation in the gene encoding the repressors Pip. To solve these problems, we (i) constructed a new integrative vector in which the gene encoding the integrase was deleted to increase its stability, and (ii) developed a new integrative vector carrying the gene encoding Pip to introduce a second copy of this gene in the chromosome. The use of these new tools was shown to reduce drastically the selection of escape mutants. |
| Databáze: | OpenAIRE |
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