Figure 1

A. Gene knockout strategy in Halobacterium sp. NRC-1. In this approach, a targeted gene allele, shown here as a deletion, is first cloned into the suicide plasmid, pBB400, which is capable of replication in E. coli (but not in Halobacterium). The plasmid also contains the native ura 3 gene under the control of its own promoter. The resulting plasmid is introduced into a Halobacterium sp. NRC-1Δura 3 host via transformation. Integrants are then selected by uracil prototrophy (Ura⁺) using commercially available uracil-dropout media components (HURA⁺ media). Subsequently, plasmid excisants are selected via counterselection of ura 3, 5-Foa-resistance (Foa^r). This gives rise to derivatives containing either the original or mutant allele, which may be distinguishable by PCR or phenotypic analysis. B. A method for construction of chromosomal knockouts of essential genes. A complementation strategy is shown where an autonomously replicating plasmid vector which contains a functional gene of interest, geneX, is introduced into the host strain, e.g. by selection for mevinolin resistance (Mev^r). Strains containing a knockout of the chromosomal copy may then be selected using the method described in part a, with the additional selection for the complementing plasmid with Mev^r.