Homologous gene knockout in the archaeon Halobacterium salinarum with ura3 as a counterselectable marker

• DOI: 10.1046/j.1365-2958.2000.01739.x
• Publication Date: 2003-03-12T11:28:37Z
• Authors: Ronald F. Peck; Shiladitya DasSarma; Mark P. Krebs
• Keywords: URA3; Auxotrophy

To facilitate the functional genomic analysis of an archaeon, we have developed a homologous gene replacement strategy for Halobacterium salinarum based on ura3 , which encodes pyrimidine biosynthetic enzyme orotidine‐5′‐monophosphate decarboxylase. H. was shown to be sensitive 5‐fluoroorotic acid (5‐FOA), can select mutations in . A spontaneous 5‐FOA‐resistant mutant found contain insertion and uracil auxotroph. Integration at bacterioopsin locus ( bop ) this restored 5‐FOA sensitivity prototrophy. Parallel results were obtained with Δ strain constructed by derivatives replaced These show that demonstrate ‐based replacement, transforming host deletion plasmid containing mevinolin resistance marker. In one approach, contained intact chromosomal locus; another, included plasmid. Plasmid integrants selected resolved 5‐FOA, yielding recombinants frequency > 10 −2 both approaches. studies establish efficient new genetic towards systematic knockout genes archaeon.