title: Linear DNA for rapid prototyping of synthetic biological circuits in an Escherichia coli based TX-TL cell-free system.
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.
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology , Pasadena, California 91101, United States of America.
authors.forename: Zachary Z
authors.initials: ZZ
authors.lastname: Sun
authors.forename: Enoch
authors.initials: E
authors.lastname: Yeung
authors.forename: Clarmyra A
authors.initials: CA
authors.lastname: Hayes
authors.forename: Vincent
authors.initials: V
authors.lastname: Noireaux
authors.forename: Richard M
authors.initials: RM
authors.lastname: Murray
mesh_terms: D002474:Cell-Free System / Q000737:chemistry*; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA; D058615:Synthetic Biology; D014158:Transcription, Genetic
chemical_list: D004247:DNA