Figure 4.
Antisense sequestration of gRNA partially restores the dynamic range of a 2× inverter. (A) Two variants of the 2× inverter controlled to have the same compositional context. For simplicity, the additional asRNA Dud node upstream of the circuit is not depicted. (B) Antisense sequestration of gRNA of the 2× inverter (yellow) partially restores the dynamic range by expanding the range of expression in both the ON and OFF states compared to the basic 2× inverter with no sequestration (blue). In linear space, the displayed error bars are ±1 standard deviation from threefold biological replicates. Performance is compared to the same GFP control (dotted green) used in Figure 3. Due to the extremely slow equilibration time of the 2× inverter, these constructs were run for additional time in order to be allowed to reach equilibrium (see Materials and Methods). (C) The same constructs, this time under the addition and subtraction of aTc in a microfluidic chamber. The presence of antisense sequestration speeds circuit response under aTc addition and removal with respect to the basic 2× inverter. Traces show the median intensities of single cells across all microfluidic channels. Shaded regions indicate ± quartiles. (D) Changes in inverter fold change (calculated as the maximum:minimum ratio of the Hill function fit) as functions of time. The indicated times (t1 and t2) correspond to measurements during the late exponential phase (see Figures S2 and S5) and stationary phase (Figures 3B and 4B), respectively.