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

Zachary Z. Sun, Enoch Yeung, Clarmyra A. Hayes, Vincent Noireaux, Richard M. Murray

Research output: Contribution to journalArticlepeer-review

125 Scopus citations

Abstract

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 enabled the use of linear DNA PCR products in TX-TL. We then compared expression levels and binding dynamics of different promoters on linear DNA and plasmid DNA. We also demonstrated assembly technology to rapidly build circuits entirely in vitro from separate parts. Using this strategy, we prototyped a four component genetic switch in under 8 h entirely in vitro. Rapid in vitro assembly has future applications for prototyping multiple component circuits if combined with predictive computational models.

Original languageEnglish (US)
Pages (from-to)387-397
Number of pages11
JournalACS Synthetic Biology
Volume3
Issue number6
DOIs
StatePublished - Jun 20 2014

Keywords

  • Golden Gate assembly
  • TX-TL
  • biomolecular breadboard
  • cell-free expression
  • rapid linear DNA assembly
  • rapid prototyping
  • synthetic biology
  • synthetic gene circuits

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