Cell-Free Gene Circuit Characterization Using High-Throughput Microfluidics

We present a microfluidic platform for high-throughput characterization of cell-free gene circuits. The device is capable of running 768 reactions in parallel, each programmed independently with PCR-generated linear DNA templates operating in E. coli lysate. We deployed the device to engineer a library of synthetic zinc finger repressor-promoter pairs, which was characterised by measuring repression, leak, and orthogonality. We designed cooperativity into the repressors by introducing dimerization domains, and used the resulting nonlinear response to build libraries of logical NAND, AND, and OR gates. The quantitative reproducibility of our microfluidic approach allowed us to describe the observed circuit behaviour with simple thermodynamic models, as well as to extract sensible biophysical parameters describing our circuit components.