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Abstract Synthetic genetic circuits offer the potential to wield computational control over biology, but their complexity is limited by the accuracy of mathematical models. Here, we present advances that enable the complete encoding of an electronic chip in the DNA carried by Escherichia coli (E. coli). The chip is a binary‐coded digit (BCD) to 7‐segment decoder, associated with clocks and calculators, to turn on segments to visualize 0–9. Design automation is used to build seven strains, each of which contains a circuit with up to 12 repressors and two activators (totaling 63 regulators and 76,000 bp DNA). The inputs to each circuit represent the digit to be displayed (encoded in binary by four molecules), and output is the segment state, reported as fluorescence. Implementation requires an advanced gate model that captures dynamics, promoter interference, and a measure of total power usage (RNAP flux). This project is an exemplar of design automation pushing engineering beyond that achievable “by hand”, essential for realizing the potential of biology.

Abstract Genetic circuits implement computational operations within a cell. Debugging them is difficult because their function is defined by multiple states (e.g., combinations of inputs) that vary in time. Here, we develop RNA‐seq methods that enable the simultaneous measurement of: (i) the states of internal gates, (ii) part performance (promoters, insulators, terminators), and (iii) impact on host gene expression. This is applied to a three‐input one‐output circuit consisting of three sensors, five NOR/NOT gates, and 46 genetic parts. Transcription profiles are obtained for all eight combinations of inputs, from which biophysical models can extract part activities and the response functions of sensors and gates. Various unexpected failure modes are identified, including cryptic antisense promoters, terminator failure, and a sensor malfunction due to media‐induced changes in host gene expression. This can guide the selection of new parts to fix these problems, which we demonstrate by using a bidirectional terminator to disrupt observed antisense transcription. This work introduces RNA‐seq as a powerful method for circuit characterization and debugging that overcomes the limitations of fluorescent reporters and scales to large systems composed of many parts.

Abstract Genetic circuits require many regulatory parts in order to implement signal processing or execute algorithms in cells. A potentially scalable approach is to use dCas9, which employs small guide RNAs (sgRNAs) to repress genetic loci via the programmability of RNA:DNA base pairing. To this end, we use dCas9 and designed sgRNAs to build transcriptional logic gates and connect them to perform computation in living cells. We constructed a set of NOT gates by designing five synthetic Escherichia coli σ70 promoters that are repressed by corresponding sgRNAs, and these interactions do not exhibit crosstalk between each other. These sgRNAs exhibit high on‐target repression (56‐ to 440‐fold) and negligible off‐target interactions (