Figure 1.

Design of a recombinase-based controller. (A) Schematic of the synthetic gene circuit, where GFP is driven by a constitutive promoter and RFP is controlled by an inducible promoter. The orientation of the inducible promoter is regulated by recombinase elements, integrase, and excisionase. Excisionase, co-expressed bicistronically with RFP, forms a complex with constitutively expressed integrase to flip the promoter by converting attL and attR sites into attB and attP sites, respectively. Integrase alone reverses this process, restoring the original promoter orientation. Re-NF-Controller and Re-NF-FF-Controller are distinguished by whether the flipped promoter drives GFP expression, with only the latter inducing GFP upon promoter inversion. (B) The extent of resource coupling is assessed by examining the relative expression of RFP and GFP. A negative correlation (blue line) indicates resource competition-driven coupling, whereas a flat relationship (red line) represents an ideal case where the controller effectively eliminates resource competition.