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Uncovering cell-free protein expression dynamics by a promoter library with diverse strengths

View ORCID ProfileSabrina Galiñanes Reyes, Yutetsu Kuruma, View ORCID ProfileSoichiro Tsuda
doi: https://doi.org/10.1101/214593
Sabrina Galiñanes Reyes
1WestCHEM, School of Chemistry, The University of Glasgow, Glasgow G12 8QQ, UK
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  • ORCID record for Sabrina Galiñanes Reyes
Yutetsu Kuruma
2Earth-Life Science Institute, Tokyo Institute of Technology, Tokyo, Japan
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Soichiro Tsuda
1WestCHEM, School of Chemistry, The University of Glasgow, Glasgow G12 8QQ, UK
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  • ORCID record for Soichiro Tsuda
  • For correspondence: tsuda@chem.gla.ac.uk
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Abstract

Cell-free protein expression systems have been widely used for synthetic biology and metabolic engineering applications in recent years. Yet little is known about protein expression in the cell-free systems. Here we take a systems approach to uncover underlying dynamics of cell-free protein expression. We construct a set of T7 promoter variants to express proteins at different transcription rates in a reconstituted and E. coli extract-based cell-free systems. We find that the maximum expression level and the rate of protein synthesis as responses to the transcription rate change are different in the two cell-free systems, suggesting they are driven by different expression dynamics. We confirm this by constructing a simple mathematical model for each cell-free system, which well reproduce the experimental results and also identify different limiting factors for better protein expression in the two cell-free systems. In particular, they revealed there is a negative feedback effect in the mRNA-protein translation by the PURE system and also identified different limiting factors for better protein expression in the two cell-free systems.

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Posted November 10, 2017.
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Uncovering cell-free protein expression dynamics by a promoter library with diverse strengths
Sabrina Galiñanes Reyes, Yutetsu Kuruma, Soichiro Tsuda
bioRxiv 214593; doi: https://doi.org/10.1101/214593
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Uncovering cell-free protein expression dynamics by a promoter library with diverse strengths
Sabrina Galiñanes Reyes, Yutetsu Kuruma, Soichiro Tsuda
bioRxiv 214593; doi: https://doi.org/10.1101/214593

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