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

Figure 4

Schematic of the multiple gene knockout system with one-step PCR (MONSTER). (a) Construction of a DNA photolyase-encoding gene (phr) deletion mutant. A plasmid-borne pyrE-lacS marker served as the PCR template, which attached S. acidocaldarius chromosomal sequences (5, 3, and partial sequences of phr at the 5 ends of the primers) to the ends of the selectable dual marker. After one-step construction, the MONSTER-phr was electroporated into strain SK-1. A double crossover between the MONSTER-phr and the chromosome at the 5 and Tg regions results in the pyrE-lacS marker and 3 region insertion at the phr locus. The resulting uracil prototroph transformants that exhibit blue colonies can be selected on uracil-free plates. A DNA photolyase deletion mutant with the marker removed was generated by pop-out recombination at two duplicated 3 regions, which can be selected by 5-FOA counterselection in combination with X-gal staining. Arrows show the positions of PCR primer sets. (b) Uracil and blue selection plate. (c) 5-FOA and white selection plate. (d) PCR analysis of the phr locus of the S. acidocaldarius strains SK-1 (ΔpyrE ΔsuaI), DP-1 Int (intermediate), and DP-1 (ΔpyrE ΔsuaI Δphr) using phr-out-F/R as primers. The expected sizes of the PCR bands were 1.5 kb (wt), 4 kb (recombinant), and 0.2 kb (deletion mutant). A λ-EcoT14 or 100 bp DNA ladder was loaded in lane M.

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