Resource usage and gene circuit performance characterization in a cell-free ‘breadboard’

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Dan Siegal-Gaskins, Zoltan A. Tuza, Jongmin Kim, Vincent Noireaux, Richard M. Murray
ACS Synthetic Biology, 3(6):416–425, 2014

The many successes of synthetic biology have come in a manner largely different from those in other en- gineering 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 benefitsÂin cost savings and design cycle time of a more traditional engineering approach can be significant. We have recently developed an in vitro Âbreadboard 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 the cell-free transcription-translation breadboard makes it a promising tool for rapid biocircuit design and testing, as well as for probing the fundamentals of gene circuit functions that are normally masked by cellular complex- ity. 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 and nucleoside triphosphate concentrations on biocircuits properties, and we isolate contributions of the essential componentsÂcore RNA polymerase, housekeeping sigma factor, and ribosomesÂto overall performance. Importantly, we show how limits on essential resources, particularly those involved in translation steps, manifest themselves in the form of reduced expression in the presence of orthogonal genes as load processes.

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