A T7 RNA polymerase-based toolkit for the concerted expression of clustered genes
Bacterial genes whose enzymes are either assembled into complex multi-domain proteins or form biosynthetic pathways are frequently organized within large chromosomal clusters. The functional expression of clustered genes, however, remains challenging since it generally requires an expression system...
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| Hauptverfasser: | , |
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| Dokumenttyp: | Article (Journal) |
| Sprache: | Englisch |
| Veröffentlicht: |
20 January 2012
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| In: |
Journal of biotechnology
Year: 2012, Jahrgang: 159, Heft: 3, Pages: 162-171 |
| ISSN: | 1873-4863 |
| DOI: | 10.1016/j.jbiotec.2012.01.004 |
| Online-Zugang: | Verlag, Volltext: http://dx.doi.org/10.1016/j.jbiotec.2012.01.004
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| Verfasserangaben: | Solmaz Arvani, Annette Markert, Anita Loeschcke, Karl-Erich Jaeger, Thomas Drepper |
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| 520 | |a Bacterial genes whose enzymes are either assembled into complex multi-domain proteins or form biosynthetic pathways are frequently organized within large chromosomal clusters. The functional expression of clustered genes, however, remains challenging since it generally requires an expression system that facilitates the coordinated transcription of numerous genes irrespective of their natural promoters and terminators. Here, we report on the development of a novel expression system that is particularly suitable for the homologous expression of multiple genes organized in a contiguous cluster. The new expression toolkit consists of an Ω interposon cassette carrying a T7 RNA polymerase specific promoter which is designed for promoter tagging of clustered genes and a small set of broad-host-range plasmids providing the respective polymerase in different bacteria. The uptake hydrogenase gene locus of the photosynthetic non-sulfur purple bacterium Rhodobacter capsulatus which consists of 16 genes was used as an example to demonstrate functional expression only by T7 RNA polymerase but not by bacterial RNA polymerase. Our findings clearly indicate that due to its unique properties T7 RNA polymerase can be applied for overexpression of large and complex bacterial gene regions. | ||
| 650 | 4 | |a Bacteriophage T7 | |
| 650 | 4 | |a Cloning, Molecular | |
| 650 | 4 | |a DNA-Directed RNA Polymerases | |
| 650 | 4 | |a Escherichia coli | |
| 650 | 4 | |a Genes, Bacterial | |
| 650 | 4 | |a Genetic Engineering | |
| 650 | 4 | |a Hydrogenase | |
| 650 | 4 | |a Models, Genetic | |
| 650 | 4 | |a Multigene Family | |
| 650 | 4 | |a Promoter Regions, Genetic | |
| 650 | 4 | |a Pseudomonas putida | |
| 650 | 4 | |a Recombinant Proteins | |
| 650 | 4 | |a Rhodobacter capsulatus | |
| 650 | 4 | |a Viral Proteins | |
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