Establishing a Markerless Genetic Exchange System for Methanosarcina mazei Strain Gö1 for Constructing Chromosomal Mutants of Small RNA Genes

Figure 2

Verification of ΔsRNA₁₅₄ strains by PCR. (a) Cartoon of plasmid pRS623 integration into the M. mazei chromosome. Primers for PCR verification are shown by arrows (sizes do not correspond to actual proportions). (b) M. mazei wild-type and mutant strains ΔsRNA₁₅₄, Δhpt, and a diploid strain with pRS632 integrated into the chromosome were tested by PCR analysis for presence of the sRNA₁₅₄ gene and ΔsRNA₁₅₄ deletion construct. Lane M, 100 bp marker, Fermentas; lane 1: M. mazei wild type; lane 2: pRS623 carrying ΔsRNA₁₅₄ deletion; lane 3: diploid strain; lanes 4–11: different ΔsRNA₁₅₄ candidate strains, whereas only the ΔsRNA₁₅₄ clone in lane 11 shows the unique genotype and was further analyzed by PCR analysis. (c) M. mazei wild-type and the same mutant strains were analyzed for presence of pRS632 backbone by PCR using primers for amplifying parts of the bla and pac genes, respectively. Lane M, 100 bp marker (Fermentas); lane 1: M. mazei WT; lane 2: Δhpt; lane 3: diploid strain; lane 4: Δ154. lane 5: pRS632; lane 6: water control.

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