Journal: ACS synthetic biology

Article Title: Modularity of select riboswitch expression platforms enables facile engineering of novel genetic regulatory devices

doi: 10.1021/sb4000096

Figure Lengend Snippet: (a) 2-aminopurine (2AP) dependent regulation of gfpuv expression by the xpt(C74U)/metE chimera in E. coli. Cells transformed with either the control parental vector (pBR322) that does not contain gfpuv, the xpt(C74U)/metE riboswitch in the 5'-leader of an mRNA encoding gfpuv, and a mutant of this riboswitch (U51C) that specifically abrogates 2AP binding to the aptamer were plated on defined medium-agar in the absence (left) or presence (right) of 1 mM 2AP. (b) Quantification of the fluorescence of E. coli transformed with the xpt(C74U)/metE chimera (grey circles) or the mutant (U51C, black squares) grown in defined medium with increasing concentrations of 2AP. (c) Theophylline-dependent control of gfpuv expression in E. coli. Cells were grown under the same media conditions as in (a), but supplemented with 1 mM theophylline in the right plate. A single point mutation in the theophylline aptamer (U24A) serves a control for potential theophylline-dependent effects on gfpuv expression not directly related to its binding to the aptamer. (d) Quantification of the fluorescence of E. coli transformed with the theo/metE chimera (grey circles) or the point mutation thereof (black squares).

Article Snippet: Briefly, 100 ng of DNA were incubated at 37 °C for 15 minutes in 25 μl of 2× transcription buffer (140 mM Tris-HCl, pH 8.0, 140 mM NaCl, 0.2 mM EDTA, 28 μM β-mercaptoethanol and 70 mg/mL BSA), 5 μl of 25 mM MgCl 2 , 1 mCi of α-32P-ATP and 0.5 units of E. coli RNA polymerase 70 holoenzyme (Epicentre Biotechnologies) to a final volume of 35 μl.

Techniques: Expressing, Transformation Assay, Control, Plasmid Preparation, Mutagenesis, Binding Assay, Fluorescence