Figure 1.

Suicide plasmids used in the construction of a chromosomal deletion and overexpression of a homologous protein with an affinity tag in M. jannaschii. The plasmids were pBluescript II SK(+) derivatives carrying a M. jannaschii-specific selectable marker composed of the HMG-CoA reductase gene (hmgA, mj_0705) of M. jannaschii (Bult et al., 1996) driven by a putative promoter of the S-layer gene (mfs40622_1341) of Methanocaldococcus FS.406-22 (Psla) (Mehta and Baross, 2006). The marker conferred resistance to mevinolin and simvastatin in M. jannaschii. (A) The plasmid pDS210, which was used as a suicide plasmid for deleting the fsr gene, contained 500 bp long DNA segments of the upstream and downstream regions of the fsr gene of M. jannaschii (locus tag number, mj_0870). (B) The plasmid pDS210 was used for constructing a chromosomal construct for the overexpression of an F₄₂₀H₂ oxidase gene (fprA, locus tag mj_0748) with an NH₂-terminal 3xFLAG-twin Strep affinity tag in M. jannaschii. It carried a cassette composed of a putative promoter of the flagellin operon (flaB1B2, mj_0891-0892) of M. jannaschii (P_flaB1B2) fused with a 174 bp long DNA element encoding the affinity tag which was flanked by 500 bp of the upstream region of fprA (locus tag number, mj_0748) and the 500 bp of the 5′ end of fprA coding sequence. bla, beta-lactamase gene conferring resistance to penicillin derivatives in E. coli. f1 ori and ColE1ori are origins of replication of the f1 phage and ColE1 plasmid, respectively. T_sla, putative terminator region of S-layer protein gene (mfs40622_1341) of Methanocaldococcus FS.406-22. Numbers on the side of plasmid maps represent sequence coordinates.