The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology

Jonathan Garamella; Ryan Marshall; Mark Rustad; Vincent Noireaux

doi:10.1021/acssynbio.5b00296

The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology

Jonathan Garamella, Ryan Marshall, Mark Rustad, Vincent Noireaux

Physics and Astronomy (Twin Cities)

Research output: Contribution to journal › Article › peer-review

163 Scopus citations

Abstract

We report on and provide a detailed characterization of the performance and properties of a recently developed, all Escherichia coli, cell-free transcription and translation system. Gene expression is entirely based on the endogenous translation components and transcription machinery provided by an E. coli cytoplasmic extract, thus expanding the repertoire of regulatory parts to hundreds of elements. We use a powerful metabolism for ATP regeneration to achieve more than 2 mg/mL of protein synthesis in batch mode reactions, and more than 6 mg/mL in semicontinuous mode. While the strength of cell-free expression is increased by a factor of 3 on average, the output signal of simple gene circuits and the synthesis of entire bacteriophages are increased by orders of magnitude compared to previous results. Messenger RNAs and protein degradation, respectively tuned using E. coli MazF interferase and ClpXP AAA+ proteases, are characterized over a much wider range of rates than the first version of the cell-free toolbox. This system is a highly versatile cell-free platform to construct complex biological systems through the execution of DNA programs composed of synthetic and natural bacterial regulatory parts.

Original language	English (US)
Pages (from-to)	344-355
Number of pages	12
Journal	ACS Synthetic Biology
Volume	5
Issue number	4
DOIs	https://doi.org/10.1021/acssynbio.5b00296
State	Published - Apr 15 2016

Bibliographical note

Funding Information:
We thank Filippo Caschera, without whom this work would have not been possible. We are grateful to Claudia Schmidt-Dannert for technical help, Natalie Ma for discussions on MS2 phage, John Wertz at CGSC (Yale), Wei Zhang for TEM performed at the Characterization Facility at the University of Minnesota, which receives partial support from NSF through the MRSEC program. This work was supported by the Office of Naval Research award number N00014-13-1-0074, by MYcroarray as part of the Defense Advanced Research Projects Agency (DARPA) and the Army Contracting Command-Aberdeen Proving Grounds (ACC-APG) under Contract/Purchase Order number W911NF-15-P-0027, and by the Human Frontier Science Program grant number RGP0037/2015.

Keywords

biosynthesis
cell-free transcription-translation
gene circuits prototyping
minimal cell
self-assembly

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title = "The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology",

abstract = "We report on and provide a detailed characterization of the performance and properties of a recently developed, all Escherichia coli, cell-free transcription and translation system. Gene expression is entirely based on the endogenous translation components and transcription machinery provided by an E. coli cytoplasmic extract, thus expanding the repertoire of regulatory parts to hundreds of elements. We use a powerful metabolism for ATP regeneration to achieve more than 2 mg/mL of protein synthesis in batch mode reactions, and more than 6 mg/mL in semicontinuous mode. While the strength of cell-free expression is increased by a factor of 3 on average, the output signal of simple gene circuits and the synthesis of entire bacteriophages are increased by orders of magnitude compared to previous results. Messenger RNAs and protein degradation, respectively tuned using E. coli MazF interferase and ClpXP AAA+ proteases, are characterized over a much wider range of rates than the first version of the cell-free toolbox. This system is a highly versatile cell-free platform to construct complex biological systems through the execution of DNA programs composed of synthetic and natural bacterial regulatory parts.",

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