Characterization of recombinase-based genetic parts and circuits using nanopore sequencing

Abstract Recombinases are versatile enzymes able to perform the precise insertion, deletion, and rearrangement of DNA and can act as a foundation for programmable genetic logic and memory. Crucial for their use are accurate measurements of function. However, these are often laborious, time-consuming, and costly to collect. To address this, we develop a semi-automated workflow that combines low-cost liquid handling robotics, multiplexed long-read nanopore sequencing, and a supporting computational analysis tool to enable the high-throughput and detailed characterization of recombinase parts and circuits when used in a variety of contexts and organisms. Our approach overcomes the limitations of typically used fluorescence-based assays and is able to monitor temporal dynamics, observe structural changes at nucleotide resolution, and unravel the internal workings of complex multi-state circuits. The ability to scale up and automate genetic circuit characterization is an essential step towards more rigorous biological metrology that can support the construction of predictive models for efficiently engineering biology.