Optimization of an In Vitro Transcription/Translation System Based on Sulfolobus solfataricus Cell Lysate

Figure 2

In vitro transcription of plasmids containing the 16S/23S rRNA promoter. (a) Schematic representation of the pBS-rRNA_p construct. Horizontal arrows indicate the position of primers used for RT-PCR analysis. (b) RT-PCR on total RNA extracted from S30 of S. solfataricus previously incubated with 2, 4 and 8 μg of pBS-rRNA_p plasmid and corresponding, respectively, to the lanes 4, 6 and 8 of the image. The product of the reaction is shown by the amplified fragment of 346 bp. Also shown is the RT-PCR of an mRNA encoding the translation factor aIF1A, used as an endogenous control to normalize the reactions. (c) Schematic representation of the pBS-rRNA_p-104 plasmid. The SD motif is evidenced in italic, while the start codon is shown in bold. (d) Relative amount of RNA transcribed by the pBS-rRNA_p-104 plasmid incubated into S. solfataricus S30 extract at 70°C for 1 h. (e) Absolute quantification of the pBS-rRNA_p-104 transcript using the standard curve method. The absolute quantities of the standards were obtained measuring the concentration of T7 in vitro-transcribed pBS-rRNA_p-104 RNA. Serial dilutions of the in vitro transcript were obtained and their Ct values (red dots) were compared to those unknown (blue dots) extrapolating the amount of copies expressed.

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