Fig. 4: Design of a modular scaffold for 3-input history-dependent programs.

3-input history-dependent scaffold (a) and its lineage tree (b). Integrase sites are positioned to permit expression of an output gene in various states of the lineage tree. For each state of the desired lineage, a different gene is expressed, and a gene is also expressed when no input is present. The four columns of the lineage tree correspond to different numbers of inputs that have occurred sequentially (from 0 to 3 inputs) and the six lineages correspond to different order of occurrences of inputs (example: a–b–c for lineage 1 and b–a–c for lineage 3). c DNA and gene-expression states of the scaffold. The gene at the GOI position 0 is expressed only when no input is present. The scaffold has six different DNA states. d Optimization of the 3-input scaffold using OSIRiS. For a given 3-input program, a scaffold with consecutive expressions of BFP, RFP, and GFP in one lineage is designed. From this design, six intermediate DNA states are generated and the expected phenotype for each DNA state of the tree is predicted. Two versions of this 3-input scaffold were analyzed. Version 1 was producing unexpected GFP fluorescence in state 0 and state 4. Version 2 is an optimized design from version 1, in which two DNA sequences corresponding to spacers sp7 and sp6, flanking L3S2P21 and J61048 terminators, were removed. The fluorescence intensities in different channels for two versions in DNA states 0 and 4 is shown. The bar graph corresponds to the fold change over the negative control (strain without fluorescent protein) for each channel (GFP, RFP, and BFP) from three experiments with three replicates per experiment. The error bars correspond to the standard deviation between the fold changes obtained in three separate experiments.