Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Dec;2(3):145-9.
doi: 10.1155/2008/948014.

Conditions for gene disruption by homologous recombination of exogenous DNA into the Sulfolobus solfataricus genome

Sonja-Verena Albers  1 , Arnold J M Driessen
Affiliations

Conditions for gene disruption by homologous recombination of exogenous DNA into the Sulfolobus solfataricus genome

Sonja-Verena Albers et al. Archaea. 2008 Dec.

Abstract

The construction of directed gene deletion mutants is an essential tool in molecular biology that allows functional studies on the role of genes in their natural environment. For hyperthermophilic archaea, it has been difficult to obtain a reliable system to construct such mutants. However, during the past years, systems have been developed for Thermococcus kodakarensis and two Sulfolobus species, S. acidocaldarius and derivatives of S. solfataricus 98/2. Here we describe an optimization of the method for integration of exogenous DNA into S. solfataricus PBL 2025, an S. solfataricus 98/2 derivative, based on lactose auxotrophy that now allows for routine gene inactivation.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Schematic overview of the plasmids pET2268 and pSVA50 used for the isolation of gene deletion mutants in PBL2025. The KpnI/NcoI and BamHI/NotI restriction sites are used to clone the upstream and downstream flanking regions of the target gene(s), respectively.
Figure 2.
Figure 2.
Analysis of genomic DNA of Solfolobus solfataricus cells derived from selected blue colonies from cells electroporated with pSVA37 on tryptone plates. The PCRs were performed with genomic DNA derived from nine individual cultures and from PBL2025 control cells. The primer set used is directed toward the targeted gene flanking regions and resulted in a 2100 bp product in the wild type cells corresponding to the undisrupted gene, and a 2900 bp band for the mutant cells corresponding to the disrupted gene with an insertion of the lacS selection marker.
Figure 3.
Figure 3.
Analysis of genomic DNA of individual cultures obtained from selected blue colonies from cells electroporated with either pSVA37 or pSVA6. The left panel shows the presence of the marker gene using lacS specific primers. The right panel shows the positive identification of successful integrations using primers specific for an internal part of lacS and the 5′ flanking region of the targeted Sso0120 gene. As a positive control (C) for the lacS PCR Solfolobus solfataricus P2 genomic DNA was used, while for the PCR of the flanking region, genomic DNA of PBL2025 was used as a negative control. The DNA marker sizes are indicated.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Albers S.-V., Driessen A.J. Analysis of ATPases of putative secretion operons in the thermoacidophilic archaeon Sulfolobus solfataricus . Microbiology. 2005;151:763–773. - PubMed
    1. Albers S.-V., Jonuscheit M., Dinkelaker S., Urich T., Kletzin A., Tampe R., Driessen A.J.M., Schleper C. Production of recombinant and tagged proteins in the hyperthermophilic archaeon Sulfolobus solfataricus . Appl. Environ. Microbiol. 2006;72:102–111. - PMC - PubMed
    1. Aucelli T., Contursi P., Girfoglio M., Rossi M., Cannio R. A spreadable, non-integrative and high copy number shuttle vector for Sulfolobus solfataricus based on the genetic element pSSVx from Sulfolobus islandicus . Nucleic Acids Res. 2006;34:e114. - PMC - PubMed
    1. Bartolucci S., Rossi M., Cannio R. Characterization and functional complementation of a nonlethal deletion in the chromosome of a β-glycosidase mutant of Sulfolobus solfataricus . J. Bacteriol. 2003;185:3948–3957. - PMC - PubMed
    1. Bernander R., Poplawski A. Cell cycle characteristics of thermophilic archaea. J. Bacteriol. 1997;179:4963–4969. - PMC - PubMed

Publication types