FIG. 3.

Schematic representation of gene replacement in the WT locus of the DES5A gene with the targeting construct (C270 fragment) by homologous recombination, generating the knockout locus in the KO270 mutant. (A) The numbered arrowheads indicate the primers used. (B) Shown are the PCR amplification products from genomic DNA of WT T. thermophila and the KO270 mutant using two allele-specific primers (7 and 8) and the locus specific primer (9) indicated above the gel (Table 1). In the WT, a 1.5-kb band was visible, while in KO270 a 1.2-kb fragment corresponding to the knockout locus was amplified. Sample WT + KO270 is a mixture of the two genomic DNAs, and it was used as a control. M, markers for fragment length. (C) Shown are RNA levels in the WT and KO270 grown in cholestanol-containing cultures, sampled at 6 and 24 h, and measured by RT-PCR. DES5A indicates the fragment amplified by using primers 10 and 11, designed on the second exon of DES5A. α-TUB indicates the fragment amplified from α-tubulin cDNA using primers 12 and 13 as a control. (D) Comparison of T. thermophila WT (•) and KO270 (▪) growth rates. Both strains showed similar growth rates and final biomass yields.