Rapid in vitro engineering of 16 two-input logic gates

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Clarmyra A. Hayes, Emmanuel L.C. de los Santos, Enoch Yeung, Sean R. Sanchez, Seung Y. Lee, and Richard M. Murray
Presented, 2016 Winter q-Bio

In vitro transcription and translation systems have been used to rapidly test and debug synthetic circuits, allowing for much faster design-build-test cycles. To demonstrate the power of in vitro prototyping, we designed 16 two-input logic gates using a library of 14 linear DNA constructs. We successfully implemented all 16 gates in an E. coli cell extract prototyping environment (TXTL), going from design to functionality in less than 3 months. In separate tests, each taking less than an hour to set up and less than 8 hours to run, we were able to quickly diagnose functionality of engineered parts, including quantification of promoter leakiness and promoter and repressor strength. In subsequent short tests we determined optimal circuit component ratios and investigated component interactions, including crosstalk and resource loading. To lower the entry barrier to in vitro testing, we also created a cellphone-based fluorescent imager that can be used to measure fluorescent output of paper-based TXTL reactions. We hope to establish in vitro testing as a rapid, easily accessible tool for engineering synthetic circuits in research and education.