Figure 4.
Analysis of Synergy Data
Synergy is calculated as a ratio of the expression in the presence of both TF and amplifier activator, divided by the sum of expression with TF only and amplifier activator only. To increase accuracy, TF-only data from Figure 1 is used for calculations (Supplemental Experimental Procedures).
(A) Transactivating efficiency of PIT-RelA amplifier activator as a function of PIR distance from the TATA box. The intervening RE for the endogenous TF input coupled to the low-leakage promoter (color coded) and the sequence of a minimal promoter (minimal TATA box versus CMVMIN, inset) are compared.
(B) Fully induced expression levels from a composite promoter achieved by providing the ectopic TF input and PIT-RelA, as a function of PIR distance from the TATA box.
(C) Synergy scores for different composite promoters with PIT-RelA as amplifier activator, as a function of PIR distance from the TATA box.
(D) Schematics of the synergistic positive-feedback amplifier.
(E and F) Simulated (E) and experimental (F) responses of open- and feedback-amplified sensors to varying TF input. Comparison to an amplified loop without synergy is shown.
(G) Simulated open-loop and amplified response dependency on the binding affinity of the TF input.
(H) Simulated relationship between open- and amplified-loop responses.
(I) Experimental correlation between open- and closed-loop responses for HNF1A/B and SOX9/10 constructs in HEK Tet-On cells.
(J) Simulated off and on responses of feedback-amplified loops as a function of synergy (left) and on-to-off ratio as a function of synergy (right).
(K) Experimental on-to-off ratio of feedback-amplified sensors as a function of synergy (black dots). The straight line is a linear fit forced through zero.
In (A), (B), and (F), error bars represent SD of biological triplicates.