Figure 2. 2-input BLADE platform can produce four distinct output functions based on two inputs.

(a) 2-input BLADE template on one plasmid with a single transcriptional unit. This template contains four distinct regions of DNA (addresses) downstream of a promoter. Each address corresponds to an output function and is accessed or deleted via site-specific DNA recombination. Each address can be programmed from different configurations ranging from zero-inputs to Boolean functions. The first address (Z₀₀), which is the closest to the promoter, corresponds to a state where no recombinase is expressed (A = 0, B = 0). If the Z₀₀ address contains a protein coding sequence, then that gene will be expressed. Gene expression from the other addresses downstream of Z₀₀ will be blocked by the presence of Z₀₀ protein coding region. In the presence of recombinase A, which corresponds to state (A = 1, B = 0), addresses Z₀₀ and Z₀₁ will be removed, thus moving address Z₁₀ directly downstream of the promoter and allowing gene expression of address Z₁₀ only to occur. Similarly, when only recombinase B is present (A = 0, B = 1), addresses Z₀₀ and Z₁₀ are excised, allowing Z₀₁ to be moved directly downstream of the promoter. Finally, when both recombinases are expressed (A = 1, B = 1), addresses Z₀₀, Z₀₁, Z₁₀ are all excised, thus placing Z₁₁ downstream of the promoter unobstructed by the other addresses. (b) Integrated 2-input BLADE decoder with tagBFP, EGFP, iRFP720, and mRuby2 as addresses Z₀₀, Z₁₀, Z₀₁, and Z₁₁ respectively. Plasmids constitutively expressing Cre and/or Flp are then stably integrated in. Three days of doxycycline (DOX) treatment is used to permit the rtTA-VP48 protein to bind to the tetracycline response elements promoter (pTRE) to activate gene expression. Mean fluorescence intensity (MFI) is plotted of either n = 1 or n = 2 independent integrations. a.u. = arbitrary units.