Control of Gene Expression in E. coli with the Twister Sister Ribozyme

In the current study, the use of the recently discovered twister sister ribozyme was analyzed to determine its ability to control gene expression in Escherichia coli. Primer extension PCR was used to create a hammerhead ribozyme that could be introduced through the P1 stem. An anti-ribosomal binding site (RBS) sequence was also included in the ribozyme construct, and the ribozyme was inserted upstream of the gfp gene cloned into the pET28a(+) plasmid. Additional ribozyme constructs inserted through the P3, P4, and P5 ribozyme stems were also constructed. Plasmids were then transformed into E. coli BL21(DE3), along with controls of pET28a(+) only and pET28a-gfp without the ribozyme, and cells were grown at 37°C for 16 h. While the pET28a-gfp control produced a high level of fluorescence, all ribozyme constructs reduced fluorescence of the gfp to near background levels, indicating a reduction in gene expression. Because the twister-sister ribozyme is Mg2+-dependent, we also tested the effect of varying levels of MgSO4 in the growth medium. In vitro transcription and activity assays demonstrated that the twister sister ribozyme constructs created in this study do possess catalytic activity, indicating that they may be of use in controlling gene expression in vivo.