title: Gene circuit performance characterization and resource usage in a cell-free "breadboard".
abstract: The many successes of synthetic biology have come in a manner largely different from those in other engineering disciplines; in particular, without well-characterized and simplified prototyping environments to play a role analogous to wind-tunnels in aerodynamics and breadboards in electrical engineering. However, as the complexity of synthetic circuits increases, the benefits--in cost savings and design cycle time--of a more traditional engineering approach can be significant. We have recently developed an in vitro "breadboard" prototyping platform based on E. coli cell extract that allows biocircuits to operate in an environment considerably simpler than, but functionally similar to, in vivo. The simplicity of this system makes it a promising tool for rapid biocircuit design and testing, as well as for probing fundamental aspects of gene circuit operation normally masked by cellular complexity. In this work, we characterize the cell-free breadboard using real-time and simultaneous measurements of transcriptional and translational activities of a small set of reporter genes and a transcriptional activation cascade. We determine the effects of promoter strength, gene concentration, and nucleoside triphosphate concentration on biocircuit properties, and we isolate the specific contributions of essential biomolecular resources-core RNA polymerase and ribosomes-to overall performance. Importantly, we show how limits on resources, particularly those involved in translation, are manifested as reduced expression in the presence of orthogonal genes that serve as additional loads on the system.
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology , Pasadena, California 91125, United States.
authors.forename: Dan
authors.initials: D
authors.lastname: Siegal-Gaskins
authors.forename: Zoltan A
authors.initials: ZA
authors.lastname: Tuza
authors.forename: Jongmin
authors.initials: J
authors.lastname: Kim
authors.forename: Vincent
authors.initials: V
authors.lastname: Noireaux
authors.forename: Richard M
authors.initials: RM
authors.lastname: Murray
mesh_terms: D000595:Amino Acid Sequence; D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics; D002474:Cell-Free System; D004247:DNA / Q000737:chemistry / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D053263:Gene Regulatory Networks; D017930:Genes, Reporter; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D012270:Ribosomes / Q000235:genetics; D058615:Synthetic Biology; D014158:Transcription, Genetic; D015533:Transcriptional Activation
chemical_list: D052157:Aptamers, Nucleotide; D012333:RNA, Messenger; D004247:DNA; D012321:DNA-Directed RNA Polymerases