FIG. 2.

Plasmids used in this study. Plasmid pJK44 carries the M. barkeri strain Fusaro ileS gene in Methanosarcina-E. coli shuttle vector pWM321 (29). The origin of replication from plasmid R6K (oriR6K) allows plasmid replication in E. coli, and the pC2A replicon allows replication in the genus Methanosarcina. The pac cassette confers puromycin resistance upon methanoarchaea, and the β-lactamase gene bla encodes resistance to ampicillin on E. coli. Plasmid pPB12 is identical to pJK44, except that it carries the ileS12 allele in the place of wild-type ileS. Plasmid pPB18 differs from pPB12 by lacking the pC2A replicon and therefore is incapable of replication in Methanosarcina. Plasmid pPB32 is a Methanosarcina-E. coli shuttle vector that carries ileS12, lacZα for blue-white screening of recombinant clones, and a multiple cloning site with numerous unique sites. Plasmids pPB31, pPB33, pPB34, and pPB35 are similar to pPB32 and are described in the text. The promoter (pmcrB) and terminator (tmcr) of the Methanococcus voltae methyl reductase operon regulate expression of the puromycin acetyltransferase (pac) gene from Streptomyces alboniger in methanoarchaea.