Fig. 1: The history-dependent integrase circuit switches between fluorescent states based on the order of inputs.

a Schematic of the integrase target circuit. The target consists of two sets of integrase sites for the PhiC31 and Bxb1 integrases; three genes for fluorescent proteins (mtagBFP2 (BFP), mCherry (RFP), NeonGreen (GFP)); and a p35S strong constitutive promoter for fluorescent expression. The initial state of the target (State 0) results in the expression of mtagBFP2. Upon expression of PhiC31, an inversion causes the target to switch to State 1 and results in the expression of RFP. Subsequent Bxb1 expression causes another inversion, flipping the promoter direction and mediating the switch to State 2, resulting in GFP expression. Alternatively, the addition of Bxb1 in a cell with the State 0 target results in a DNA excision, causing a switch to State α and loss of fluorescence. b The PhiC31-Bxb1 integrase order mediates a series of switches from State 0 to 1 to 2. (left) The State 0 target shows strong BFP expression in the leaf and the root. (middle) Constitutive PhiC31 expression mediates a switch of the target to State 1 and strong RFP fluorescence in leaf and root tissue. (right) Subsequent constitutive expression of Bxb1 mediates a switch from State 1 to 2 and results in GFP expression in the leaf and root. c Expression of Bxb1 prior to PhiC31 mediates an ‘out of order’ switch to State α and loss of fluorescence. (left) The initial target in State 0 shows strong BFP expression in the leaf and the root. (right) Constitutive expression of Bxb1 with the target in State 0 results in a switch to State α and a loss in fluorescence in the leaf and root. The root and leaf tissue of n = 10 seedlings from at least 2 T1 lines were characterized for each of the target States 0, 1, 2, and α. Black scale bars in the brightfield images correspond to 100 μm (root) and 50 μm (leaf). Source data are provided as a Source Data file.