Figure 5.

gRNA switches can be triggered by the araB mRNA in TXTL and in E. coli. (A) Sensing the mRNA encoded by araBAD to drive deGFP silencing with gRNA switches. (B) Assessing gRNA switches designed to sense the araB mRNA in TXTL. See Figure 2C for details. Locations of the trigger RNAs within the araB mRNA are shown in Supplementary Figure S8. Some of the gRNA switches exhibited increased fluorescence with the araB mRNA trigger, possibly due to differences in the size and sequence of the expressed transcripts (70). (C) Assessing activation of gRNA switches in E. coli when constitutively expressing the araB mRNA or a lacZ mRNA control. The endogenous araBAD operon was deleted to prevent switch activation from this locus. Growth and fluorescence measurements were made on a microtiter plate reader. GFP expression rates were calculated as differentials of fluorescence over ABS₆₀₀ and normalized to the non-targeting (0% GFP expression rate) and targeting gRNA (100% GFP expression rate). Sw-1 with its cognate trigger (Tr-1) or a randomized RNA trigger (Rdm) served as controls. Values represent the average and standard deviation of three independent experiments initiated from separate colonies. (D) Assessing activation of gRNA switches when inducing expression of the endogenous araBAD operon. E. coli cells were induced with the addition of L-arabinose or repressed with the addition of D-glucose to the medium. See C for details.