Design, Fabrication, and Device Chemistry of a 3-Input-3-Output Synthetic Genetic Combinatorial Logic Circuit with a 3-Input AND Gate in a Single Bacterial Cell.

Advancement of in-cell molecular computation requires multi-input-multi-output genetic logic devices. However, increased physical size, a higher number of molecular interactions, cross-talk, and complex systems level device chemistry limited the realization of such multi-input-multi-output devices in a single bacterial cell. Here, by adapting a circuit minimization and conjugated promoter engineering approach, we created the first 3-input-3-output logic function in a single bacterial cell. The circuit integrated three extracellular chemical signals as inputs and produced three different fluorescent proteins as outputs following the truth table of the circuit. First, we created a noncascaded 1-gate-3-input synthetic genetic AND gate in bacteria. We showed that the 3-input AND gate was digital in nature and mathematically predictable, two important characteristics, which were not reported for previous 3-input AND gates in bacteria. Our design consists of a 128 bp DNA scaffold, which conjugated various protein-binding sites in a single piece of DNA and worked as a hybrid promoter. The scaffold was a few times smaller than the similar 3-input synthetic genetic AND gate promoter reported. Integrating this AND gate with a new 2-input-2-output integrated circuit, which was also digital-like and predictive, we created a 3-input-3-output combinatorial logic circuit. This work demonstrated the integration of a 3-input AND gate in a larger circuit and a 3-input-3-output synthetic genetic circuit, both for the first time. The work has significance in molecular computation, biorobotics, DNA nanotechnology, and synthetic biology.