TY - JOUR
T1 - The All E. coli TX-TL Toolbox 2.0
T2 - A Platform for Cell-Free Synthetic Biology
AU - Garamella, Jonathan
AU - Marshall, Ryan
AU - Rustad, Mark
AU - Noireaux, Vincent
N1 - Publisher Copyright:
© 2016 American Chemical Society.
PY - 2016/4/15
Y1 - 2016/4/15
N2 - 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.
AB - 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.
KW - biosynthesis
KW - cell-free transcription-translation
KW - gene circuits prototyping
KW - minimal cell
KW - self-assembly
UR - http://www.scopus.com/inward/record.url?scp=84966334405&partnerID=8YFLogxK
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U2 - 10.1021/acssynbio.5b00296
DO - 10.1021/acssynbio.5b00296
M3 - Article
C2 - 26818434
AN - SCOPUS:84966334405
SN - 2161-5063
VL - 5
SP - 344
EP - 355
JO - ACS Synthetic Biology
JF - ACS Synthetic Biology
IS - 4
ER -