Journal
EXTREMOPHILES
Volume 13, Issue 4, Pages 735-746Publisher
SPRINGER JAPAN KK
DOI: 10.1007/s00792-009-0254-2
Keywords
pyrEF marker; Unmarked gene deletion; S. islandicus host-vector system; pyrEF lacS deletion mutant
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Funding
- Danish Research Council of Technology and Production [274-07-0116]
- Danish Science Research Council [272-05-400]
- Huazhong Agricultural University, China
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Sulfolobus islandicus is being used as a model for studying archaeal biology, geo-biology and evolution. However, no genetic system is available for this organism. To produce an S. islandicus mutant suitable for genetic analyses, we screened for colonies with a spontaneous pyrEF mutation. One mutant was obtained containing only 233 bp of the original pyrE sequence in the mutant allele and it was used as a host to delete the beta-glycosidase (lacS) gene. Two unmarked gene deletion methods were employed, namely plasmid integration and segregation, and marker replacement and looping out, and unmarked lacS mutants were obtained by each method. A new alternative recombination mechanism, i.e., marker circularization and integration, was shown to operate in the latter method, which did not yield the designed deletion mutation. Subsequently, Sulfolobus-E. coli plasmid shuttle vectors were constructed, which genetically complemented Delta pyrEF Delta lacS mutation after transformation. Thus, a complete set of genetic tools was established for S. islandicus with pyrEF and lacS as genetic markers.
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