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. Author manuscript; available in PMC: 2018 Jun 4.
Published in final edited form as: Cell. 2017 Oct 19;171(5):1138–1150.e15. doi: 10.1016/j.cell.2017.09.049

Figure 5. Multi-output AND Gate Architecture and Performance.

Figure 5

(A) The incorporation of a synthetic transcription factor (GAD) as the output of module 1 means that it is only expressed at high levels when both P1 and P2 are high (state [1,1]). GAD enables facile tuning of downstream gene expression (mKate2 ⋯ Output N).

(B) mKate2 expression can be modulated by modifying the number of GAD BSs in the synthetic GALp expressing mKate2 (G5, G8, G14 for 5, 8, 14 GAD BSs in GALp) and the presence of miRNA BSs at the mKate2 3′ UTR (G5-Pe, G8-Pe, G14-Pe indicate transcripts with a BS[Pe]). Note the circuit diagram depicts the version with a perfect miR1 BS in the mKate2 transcript. State [0,0] indicates cells containing the negative control modules, module 1con and module 2con (see STAR Methods for details). State [0,1] indicates cells containing module 1con and module 2. State [1,0] indicates cells containing module 1 and module 2con. State [1,1] indicates cells containing module 1 and module 2. All cell states tested also contained their respective module 3.

(C) The G8-Pe AND gate architecture triggered high mKate2 expression in human ovarian cancer cells (OV8) but not in normal cells in state [1,1].

(D) Our circuit design prevents potential off-target effects in primary human T cells. The S(E2F1)p promoter was active in T cells but S(cMYC)p was inactive in T cells. The G8-Pe circuit triggered minimal mKate2 output in T cells, thus minimizing the potential off-target effects that would have been observed if the S(E2F1)p was used to directly drive mKate2 expression.

(E) Our circuit design can be readily adapted to distinguish a different tumor type from its normal counterparts. By using two different promoters, the G8-Pe circuit triggered a strong output in the breast cancer cell line MDA-MB-453 but minimal output in the non-tumorigenic breast epithelial cell line MCF-10A.

Error bars represent SEM, n = 3 biological replicates for all experiments, except n = 6 biological replicates for OV8 group in (C).

See also Figure S4A.