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. 2019 Oct 12;366(17):fnz216. doi: 10.1093/femsle/fnz216

Figure 2.

Figure 2.

sat can be used as a positive selectable marker for the construction of gene deletion mutants in M. acetivorans. (A) A schematic of M. acetivorans ΔssuC mutant generation. A linear DNA fragment containing sat flanked by 2 kilobase regions homologous to the upstream and downstream regions of ssuC was transformed into M. acetivorans. Incorporation of sat-containing DNA at the ssuC locus gives rise to mutants which are both nourseothricin and BES resistant. Primers used to verify ΔssuC::sat mutants are represented by the red arrows in the schematic. (B) PCR analysis of M. acetivorans ΔssuC mutants. Lanes 1–3 represent PCR reactions containing M. acetivorans (WWM 83; Parent strain) DNA with primers 5 and 6 (Lane 1), 6 and 7 (Lane 2) or satF and satR (Lane 3). Lanes 4–6 represent PCR reactions containing DNA from a single M. acetivorans nourseothricin resistant ssuC mutant (ΔssuC::sat) with primers 5 and 6 (Lane 4), 6 and 7 (Lane 5) or satF and satR (Lane 6). A total of 15 out of 15 independent mutants produced identical PCR products to those shown for the mutant in this figure.