Fig. 8: Composing Boolean logic.

a The cartoon summarizes a strategy for single-layer, promoter-based logic gates with ZF-TFs. We hypothesized that AND gate promoters could be designed by using multiple repeats of a paired ZF3/ZF2 motif. Full occupancy of this promoter by both ZF2a and ZF3a mimics a fully occupied x6-C promoter, and partial occupancy (with either ZFa alone) mimics an x3-S promoter. Thus, there is a large increase in gene expression when the promoter is occupied by two types of ZFa compared to one type. b Candidate two-input AND gates were constructed using one to four repeats of paired binding sites in the promoter. AND gate behavior is considered significant if reporter expression with both ZFa is greater than the sum of reporter expression with each ZFa individually (one-tailed Welch’s t-test: *p < 0.05, **p < 0.01). c Two-input dose response for the AND gate with three repeats of paired binding sites. The landscape is shaded from green to purple to facilitate visualization in the z-axis direction. d A theoretical model of COMET AND behavior is compared with other models of transcriptional AND gates; the latter vary in whether activators have multiplicative cooperativity (ρ) and whether maximum activation (α) is equivalent for TFs individually and together (Methods). e A three-input AND gate was constructed using two repeats of a triplet binding site motif. AND gate behavior is considered significant if reporter expression with all three ZFa is greater than the sum of reporter expression with each ZFa individually, and also greater than the sum from each of the three combinations with two co-expressed ZFa and the other ZFa individually (one-tailed Welch’s t-test, **p < 0.01 for all four of these tests). f A four-input gate was constructed using the binding site arrangement shown. Experiments were conducted in biologic triplicate, and data were analyzed as described in Methods. Error bars represent the S.E.M. Source data are provided in the Source Data file.