Figure 2.

Plasmids used in the study. pC2A is a naturally occurring plasmid from Methanosarcina acetivorans. The genes shown are described in the text. pJK3 is a pBluescript derivative carrying a modified pac gene cassette that confers Pur^R upon methanoarchaea. The promoter (pmcrB) and terminator (tmcr) of the Methanococcus voltae methyl reductase operon regulate expression of the puromycin acetyltransferase gene (pac) from Streptomyces alboniger in methanoarchaea. pWM307, pWM311, and pWM321 are Methanosarcina–E. coli shuttle vectors. The origin of replication from the plasmid R6K allows maintenance in E. coli, and manipulation of copy number by choice of an appropriate host strain. Each also carries the entire pC2A replicon for replication in Methanosarcina. Plasmids pWM309, pWM313, pWM315, pWM317, and pWM319 (not shown) are like pWM311 but carry different polylinkers within the lacZα gene. The lacZα gene allows blue-white screening of recombinant clones in E. coli for pWM309, pWM311, pWM313, pWM315, pWM317, and pWM319, but not in pWM321. The β-lactamase gene (bla) encodes resistance to penicillin derivatives in E. coli.