A Framework for the Modular and Combinatorial Assembly of Synthetic Gene Circuits.

Synthetic gene circuits emerge from iterative design-build-test cycles. Most commonly, the time-limiting step is the circuit construction process. Here, we present a hierarchical cloning scheme based on the widespread Gibson assembly method and make the set of constructed plasmids freely available. Our two-step modular cloning scheme allows for simple, fast, efficient, and accurate assembly of gene circuits and combinatorial circuit libraries in Escherichia coli. The first step involves Gibson assembly of transcriptional units from constituent parts into individual intermediate plasmids. In the second step, these plasmids are digested with specific sets of restriction enzymes. The resulting flanking regions have overlaps that drive a second Gibson assembly into a single plasmid to yield the final circuit. This approach substantially reduces time and sequencing costs associated with gene circuit construction and allows for modular and combinatorial assembly of circuits. We demonstrate the usefulness of our framework by assembling a CRISPR-based double-inverter circuit and a combinatorial library of 3-node networks.