Linearized pKMSW72 quantified spectrophotometically was used as the standard for qPCR quantification (Fig. S3). Sulfolobus solfataricus PH1 cell-free extract was the no template control. The qPCR settings were as follows: one cycle at 95 °C for 15 min followed by 40 cycles of 95 °C for 30 s, 60 °C for 1 min, and 72 °C for 30 s. A melting curve was determined after the last cycle to ensure that the measured fluorescence was due to the specific product. The qPCR was performed in triplicate for all samples. In order learn more to determine whether the core promoters of the 16S/23S rRNA gene (42 bp) and TF55α genes (39 bp) were sufficient for expression of the lacS reporter gene in vivo, we measured β-galactosidase activity in cell-free extracts

of S. solfataricus PH1 (lacS∷ISC1217) (Schleper et al., 1994) transformed with viral vectors containing

the respective promoter–lacS gene fusions (Fig. 2). A construct containing 200 bp upstream of lacS was used as a positive control. Cell-free extracts from transformants with all three promoters had higher levels of β-galactosidase activity than host background activity, indicating that Epigenetic inhibitor even the 39/42 bp core promoter sequences were sufficient for lacS expression in vivo (Fig. 2a). The pattern of β-galactosidase activity did not change significantly when normalized for the relative copy number of the lacS reporter gene by Southern hybridization (Fig. 2b). Previous Sulfolobus in vivo gene expression studies using similar SSV1-based reporter Molecular motor gene constructs have shown that 448 bp for the TF55α promoter (Jonuscheit et al., 2003) or 241 bp for the araS promoter (Lubelska et al., 2006) are sufficient for expression of the lacS gene. A 55-bp core promoter plus an ‘ara-box’ is sufficient for expression of lacS when in a pRN2-plasmid-based

vector, but not when the ‘ara-box’ is removed (Peng et al., 2009). To determine whether the core 16S/23S rRNA gene promoter is regulated in vivo in response to the growth phase in S. solfataricus PH1, we measured the β-galactosidase activity in S. solfataricus PH1 containing the 16S/23S rRNA gene core promoter–lacS gene fusion during lag, mid-exponential, and stationary growth phases. Similar constructs with the TF55α core and wild-type lacS promoters were tested to determine whether regulation is promoter specific. Sulfolobus solfataricus strains PH1 and P1 were included as negative and positive controls for β-galactosidase activity, respectively. The β-galactosidase activity did not change drastically between different phases of the growth cycle in wild-type S. solfataricus P1 or S. solfataricus PH1 containing the TF55αp–lacS fusion, indicating that the wild-type lacS promoter and the core TF55α promoter are not regulated with growth phase (Fig. 3). However, β-galactosidase activity produced by S. solfataricus PH1 containing the 16S/23S rRNAp gene–lacS fusion increased approximately threefold during exponential growth compared with lag phase (Fig.

No related posts.