Mu-driven transposition of recombinant mini-Mu unit DNA in the Corynebacterium glutamicum chromosome
| Autor: | Sergey Vasilievich Smirnov, Irina L. Tokmakova, Natalya V. Gorshkova, Juliya S. Lobanova, Valerii Z. Akhverdyan, Sergey V. Mashko, Alexander A. Krylov |
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| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Genetics Microbial Cointegrate Replicative transposition 030106 microbiology Genetic Vectors Corynebacterium Gene Dosage Applied Microbiology and Biotechnology Corynebacterium glutamicum Transposition (music) Bacteriophage mu 03 medical and health sciences chemistry.chemical_compound Plasmid Fluorescence proteins Intrachromosomal amplification Enhancer Excisable enhancer Gene Editing Recombination Genetic biology Methods and Protocols Cre-mediated excision General Medicine Random integration Chromosomes Bacterial biology.organism_classification Molecular biology chemistry DNA Transposable Elements Bacteriophage Mu Biotechnology Plasmids |
| Zdroj: | Applied Microbiology and Biotechnology |
| ISSN: | 1432-0614 0175-7598 |
| Popis: | A dual-component Mu-transposition system was modified for the integration/amplification of genes in Corynebacterium. The system consists of two types of plasmids: (i) a non-replicative integrative plasmid that contains the transposing mini-Mu(LR) unit bracketed by the L/R Mu ends or the mini-Mu(LER) unit, which additionally contains the enhancer element, E, and (ii) an integration helper plasmid that expresses the transposition factor genes for MuA and MuB. Efficient transposition in the C. glutamicum chromosome (≈ 2 × 10−4 per cell) occurred mainly through the replicative pathway via cointegrate formation followed by possible resolution. Optimizing the E location in the mini-Mu unit significantly increased the efficiency of Mu-driven intramolecular transposition–amplification in C. glutamicum as well as in gram-negative bacteria. The new C. glutamicum genome modification strategy that was developed allows the consequent independent integration/amplification/fixation of target genes at high copy numbers. After integration/amplification of the first mini-Mu(LER) unit in the C. glutamicum chromosome, the E-element, which is bracketed by lox-like sites, is excised by Cre-mediated fashion, thereby fixing the truncated mini-Mu(LR) unit in its position for the subsequent integration/amplification of new mini-Mu(LER) units. This strategy was demonstrated using the genes for the citrine and green fluorescent proteins, yECitrine and yEGFP, respectively. Electronic supplementary material The online version of this article (10.1007/s00253-018-8767-1) contains supplementary material, which is available to authorized users. |
| Databáze: | OpenAIRE |
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