Implementing synthetic circuit dynamics through protein degradation in an Escherichia coli based TX-TL cell-free expression system

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Clarmyra Hayes, Zachary Z. Sun, Vincent Noireaux, Richard Murray

Caltech, United States

In vitro E. coli cell-free expression systems are emerging as a powerful new platform for engineering synthetic circuits. Cell-free expression systems are unencumbered by complications inherent to living cells, such as the potential toxicity of expressed products, the cells’ drive to survive, adapt and evolve, and the extraordinary complexity of molecular crosstalk. A recently developed TX-TL cell-free expression system preserves the native E. coli transcription-translation machinery, allowing rapid debugging of novel constructs and a detailed exploration of how individual circuit elements affect circuit performance. However, extract-based systems are currently limited in their ability to probe circuit dynamics, as cell division is non-existent and protein degradation is minimal. Control of degradation further expands the complexity of circuits (e.g. oscillators) that can be tested in vitro. We improve on the TX-TL cell-free expression system by implementing a biochemical emulation of cellular division using an ATP-powered protease and specific proteolysis tags. This system allows us to finely control degradation rates in TX-TL. To demonstrate protein degradation technology, we construct a novel type 1 incoherent feed-forward loop (I1-FFL). I1-FFLs are common motifs in living cells, used to both generate a pulse-like signal and to accelerate system response time. Our I1-FFL utilizes sigma factors, a repressor and a novel combinatorial promoter to generate a pulse of fluorescent reporter protein. We conclude with current efforts to translate in vitro I1-FFLs constructed in TX-TL to in vivo.