Accelerating the design-build-test cycle of synthetic biological circuits in E. coli using S30 TX-TL cell-free systems, linear DNA, and modular assembly

Decreasing the design-build-test cycle length is a fundamental challenge facing all engineering disciplines. This is acutely true in synthetic biology, where testing of circuits is limited by the lack of an intermediary, such as a breadboard, between the end chassis (a cell) and the starting engineering point. Limitations also exist in DNA assembly and cloning, as implementation of circuits in vivorequires all parts to be localized on plasmids with compatible origins of replications. While some limitations can be overcome by massively parallel and automated methods of cloning, transformation, and testing, the ability to do so is available only at select core facilities and is currently cost-prohibitive to widely emulate.

We propose using an open-source S30-based TX-TL cell-free system we have developed to serve as a “biological breadboard” intermediate for circuits before final in vivo implementation. Using protocols designed to require minimal infrastructure investment, the cell free system is extremely portable and generalizable. A typical design-build-test cycle length in vivo without automation can take a week or more. On plasmids in TX-TL, this length can be reduced to 1-2 days. Using linear DNA and a modular assembly platform, we demonstrate a further reduction to 4-8 hours. We show the implementation of short design-build-test cycles to prototype circuits such as genetic switches, novel RNA-regulated feed-forward loops based on repression and activation, and multiple protein-based feed forward loop variants. We also demonstrate the use of TX-TL to characterize robustness of individual synthetic biology “parts,” and explore principles for transitioning results in-vitro to in-vivo.