Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius

Figure 8

Development of a transformation system based on agmatine selection for S. acidocaldarius. (a) Construction of pSAV2-argD. The S. solfataricus pyrEF operon was replaced by a 0.6 kb S. solfataricus argD marker. The resulting vector was named pSAV2-argD. (b) Transformation of the argD deletion mutant SK-5 with pSAV2-argD. Plating of SK-5 (ΔpyrE ΔsuaI ΔargD) transformed with 8 ng of plasmid DNA of pSAV2-argD (SK-5 [pSAV2-argD]) on an XTU plate at 75°C for 10 days and SK-1 (ΔpyrE ΔsuaI) transformed with 8 ng of plasmid DNA of pSAV2 (SK-1 [pSAV2]) on an XT plate at 75°C for 7 days are shown. Controls (SK-1 and SK-5) included competent cells without plasmid DNA and electroporation.

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