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

Figure 1

Schematic and optimization of transformation procedure. A marker cassette containing the 5 and 3 homologous regions of the target locus at both ends of pyrE-lacS marker genes was electroporated into strain SK-1 (ΔpyrE ΔsuaI) under various conditions: (i) DNA topology, (ii) CaCl₂ treatment, (iii) growth phase of competent cells, (iv) DNA volume, and (v) length of flanking region. After electroporation, cells were cultivated in recovery buffer (vi) as needed and plated onto XT plates by spreading or overlay plating (vii). The resulting colonies were defined as transformants and counted.