Greco, F. V. | Search bioRxiv

bioRxiv2025

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

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. Fundamental to their use are accurate measurements of function. However, these are often laborious, time-consuming, and costly to collect. To address this, we developed 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 a 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.

synthetic biology↗

bioRxiv2020

Harnessing the central dogma for stringent multi-level control of gene expression

Strictly controlled inducible gene expression is crucial when engineering biological systems where even tiny amounts of a protein have a large impact on function or host cell viability. In these cases, leaky protein production must be avoided at all costs, but ideally without affecting the achievable range of expression. Here, we demonstrate how the central dogma offers a simple way to effectively address this challenge. By simultaneously regulating both transcription and translation, we show how relative basal expression of an inducible system can be greatly reduced, with minimal impact on the maximum induced expression rate. Using this approach, we create several stringent expression systems displaying >1000-fold change in their output after induction in vivo and up to a 350-fold change when used in a cell-free expression system. Furthermore, we find that multi-level regulation is able to suppress transcriptional noise and creates a digital-like switch when transitioning between on and off states. This work provides foundational knowledge and a genetic toolkit of parts to create multi-level gene expression controllers for those working with toxic genes or requiring precise regulation and propagation of cellular signals. It also demonstrates the value of exploring more complex and diverse regulatory designs for synthetic biology.

synthetic biology↗