Fig. 2: Recombinase attachment half-site omission and design heuristics for engineering an interception synthetic memory circuit.

a Schematic of the recombinase deletion circuit, in which a reporter circuit comprised of a constitutive promoter, ribozyme, RBS, and green fluorescent protein (GFP) reading frame flanked by an aligned attB and attP pair (STATE 1). Note: We used a genetic insulator (ribozyme) to catalyze the removal of the 5’ UTR of the transcript to normalize GFP expression. A recombinase (RECOM) matched to the given att sites catalyzes recombination between attB and attP, resulting in deletion of the entire circuit (STATE 2). b–i Half-site omission tests with various recombinases are shown. For each plot, the two data bars shown in the shaded area represent controls; S1 measures fluorescence of cells transformed with the reporter plasmid alone, and S2 measures fluorescence of cells transformed with the reporter plasmid plus the corresponding constitutive recombinase expression plasmid. In the unshaded areas half-site omissions are shown with the cognate recombinases present. B1 refers to a construct in which the first half of the attB site has been omitted, B2 refers to a construct where the second half of the attB site has been omitted, likewise half-site omissions P1 and P2 correspond to positions in the attP site. Details for each recombinase are given in Supplementary Figs. 2 and 3. j A granular description of an attachment site substituted with a 16 base pair operator. In this example the O^ttg operator is substituted within an attP site that corresponds to recombinase A118. The O^ttg DNA operator is cognate to the E⁺_HQN transcription factor. k Schematic of the putative mechanism of interception; in the gray box at left, repressor binding at the O^ttg DNA operator (within the attP site) protects the circuit from deletion catalyzed by the A118 recombinase. To the right, when the repressor is induced, it unbinds from the att site, and A118 can recombine and delete the circuit. l A variety of operator positions were tested, identifying the P + 1 location as the best candidate for controlling recombination with interception. Source data are provided as a Source Data file. Data in (b–i) and (l) represent the average of n = 6 biological replicates. Error bars correspond to the SEM of these measurements.