Development of pyrF-based gene knockout systems for genome-wide manipulation of the archaea Haloferax mediterranei and Haloarcula hispanica
The haloarchaea Haloferax mediterranei and Haloarcula hispanica are both polyhydroxyalkanoate producers in the domain Archaea, and they are becoming increasingly attractive for research and biotechnology due to their unique genetic and metabolic features. To accelerate their genome-level genetic and...
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| Published in | 遗传学报 Vol. 38; no. 6; pp. 261 - 269 |
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| Main Author | |
| Format | Journal Article |
| Language | Chinese |
| Published |
2011
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| Subjects | |
| Online Access | Get full text |
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| Summary: | The haloarchaea Haloferax mediterranei and Haloarcula hispanica are both polyhydroxyalkanoate producers in the domain Archaea, and they are becoming increasingly attractive for research and biotechnology due to their unique genetic and metabolic features. To accelerate their genome-level genetic and metabolic analyses, we have developed specific and highly efficient gene knockout systems for these two haloarchaea. These gene knockout systems consist of a suicide plasmid vector with the pyrF gene as the selection marker and a uracil auxotrophic haloarchaeon （apyrF） as the host. For in-frame deletion of a target gene, the suicide plasmid carrying the flanking region of the target gene was transferred into the corresponding apyrF host. After positive selection of the single-crossover integration recombinants （pop-in） on AS-168SY medium without uracil and counterselection of the double-crossover pyrF-excised recombinants （pop-out） with 5-fluoroorotic acid （5-FOA）, the target gene knockout mutants were confirmed b |
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| Bibliography: | The haloarchaea Haloferax mediterranei and Haloarcula hispanica are both polyhydroxyalkanoate producers in the domain Archaea, and they are becoming increasingly attractive for research and biotechnology due to their unique genetic and metabolic features. To accelerate their genome-level genetic and metabolic analyses, we have developed specific and highly efficient gene knockout systems for these two haloarchaea. These gene knockout systems consist of a suicide plasmid vector with the pyrF gene as the selection marker and a uracil auxotrophic haloarchaeon （apyrF） as the host. For in-frame deletion of a target gene, the suicide plasmid carrying the flanking region of the target gene was transferred into the corresponding apyrF host. After positive selection of the single-crossover integration recombinants （pop-in） on AS-168SY medium without uracil and counterselection of the double-crossover pyrF-excised recombinants （pop-out） with 5-fluoroorotic acid （5-FOA）, the target gene knockout mutants were confirmed b |
| ISSN: | 1673-8527 |