Linear DNA for rapid prototyping of synthetic biological circuits in an Escherichia coli based TX-TL cell-free system

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Zachary Z. Sun, Enoch Yeung, Clarmyra A. Hayes, Vincent Noireaux, Richard M. Murray
Submitted, ACS Synthetic Biology, September 2013

Accelerating the pace of synthetic biology experiments requires new approaches for rapid prototyping of circuits from individual DNA regulatory elements. However, current testing standards require days to weeks due to cloning and in vivo transformation. In this work, we first characterized methods to protect linear DNA strands from exonuclease degradation in an Escherichia coli based transcription-translation cell-free system (TX-TL), as well as mechanisms of degradation. This enables the use of linear DNA PCR products in TX-TL. We then explored methods to calibrate linear DNA to plasmid DNA by concentration. We also demonstrated assembly technology to rapidly build circuits entirely in vitro from separate parts. Using this strategy, we prototyped a four-piece genetic switch in under 8 hours entirely in vitro. Rapid in vitro assembly has applications for prototyping circuits of unlimited size when combined with predictive computational models.