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Gene circuit performance characterization and resource usage in a cell-free ‘breadboard’

Dan Siegal-Gaskins, Zoltan A. Tuza, Jongmin Kim, Vincent Noireaux, Richard M. Murray
doi: https://doi.org/10.1101/000885
Dan Siegal-Gaskins
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA
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Zoltan A. Tuza
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Jongmin Kim
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Vincent Noireaux
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Richard M. Murray
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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.

Footnotes

  • ↵† Caltech Biology and Biological Engineering

  • ↵‡ PPCU

  • ↵¶ Minnesota

  • ↵§ Caltech Control and Dynamical Systems

  • ↵E-mail: dsg{at}caltech.edu

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license.
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Posted March 09, 2014.
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Gene circuit performance characterization and resource usage in a cell-free ‘breadboard’
Dan Siegal-Gaskins, Zoltan A. Tuza, Jongmin Kim, Vincent Noireaux, Richard M. Murray
bioRxiv 000885; doi: https://doi.org/10.1101/000885
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Gene circuit performance characterization and resource usage in a cell-free ‘breadboard’
Dan Siegal-Gaskins, Zoltan A. Tuza, Jongmin Kim, Vincent Noireaux, Richard M. Murray
bioRxiv 000885; doi: https://doi.org/10.1101/000885

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