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

Figure 5

In-frame deletion of argD via the MONSTER. (a) Construction of an argD 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 argD at the 5 ends of the primers) to the ends of the selectable dual marker. After one-step construction, the MONSTER-argD was electroporated into strain SK-1. A double crossover between the MONSTER-argD and the chromosome at the Tg and 3 regions results in the pyrE-lacS marker and 5 region insertion at the argD locus. The resulting uracil prototroph transformants exhibit blue colonies and can be selected on uracil-free plates. An argD deletion mutant with the marker removed was generated by pop-out recombination at two duplicated 5 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 argD locus of the S. acidocaldarius strains SK-1 (ΔpyrE ΔsuaI), SK-5 Int (intermediate), and SK-5 (ΔpyrE ΔsuaI ΔargD) using argD-F-F/R as primers. The expected sizes of the PCR bands were 0.5 kb (wt), 3 kb (recombinant), and 0.1 kb (deletion mutant). A λ-EcoT14 or 100 bp DNA ladder was loaded in lane M.

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