High-Throughput Optimization Cycle of a Cell-Free Ribosome Assembly and Protein Synthesis System

Filippo Caschera; Ashty S. Karim; Gianluca Gazzola; Anne E. D'Aquino; Norman H. Packard; Michael C. Jewett

doi:10.1021/acssynbio.8b00276

High-Throughput Optimization Cycle of a Cell-Free Ribosome Assembly and Protein Synthesis System

Filippo Caschera, Ashty S. Karim, Gianluca Gazzola, Anne E. D'Aquino, Norman H. Packard, Michael C. Jewett^*

^*Corresponding author for this work

Chemical and Biological Engineering

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

35 Scopus citations

Abstract

Building variant ribosomes offers opportunities to reveal fundamental principles underlying ribosome biogenesis and to make ribosomes with altered properties. However, cell viability limits mutations that can be made to the ribosome. To address this limitation, the in vitro integrated synthesis, assembly and translation (iSAT) method for ribosome construction from the bottom up was recently developed. Unfortunately, iSAT is complex, costly, and laborious to researchers, partially due to the high cost of reaction buffer containing over 20 components. In this study, we develop iSAT in Escherichia coli BL21Rosetta2 cell lysates, a commonly used bacterial strain, with a cost-effective poly sugar and nucleotide monophosphate-based metabolic scheme. We achieved a 10-fold increase in protein yield over our base case with an evolutionary design of experiments approach, screening 490 reaction conditions to optimize the reaction buffer. The computationally guided, cell-free, high-throughput technology presented here augments the way we approach multicomponent synthetic biology projects and efforts to repurpose ribosomes.

Original language	English (US)
Pages (from-to)	2841-2853
Number of pages	13
Journal	ACS synthetic biology
Volume	7
Issue number	12
DOIs	https://doi.org/10.1021/acssynbio.8b00276
State	Published - Oct 24 2018

Funding

This work was supported by the Army Research Office W911NF-16-1-0372 (to M.C.J.), National Science Foundation grant MCB-1716766 (to M.C.J.), the Human Frontiers Science Program RGP0015/2017 (to M.C.J.), the David and Lucile Packard Foundation (to M.C.J.), and the Camille Dreyfus Teacher-Scholar Program (to M.C.J.). A.S.K. and A.E.D. are supported by NSF Graduate Research Fellowships. The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies or endorsements, either expressed or implied, of the DoD or the U.S. Government.

Keywords

evolutionary design of experiments
iSAT
in vitro
machine learning
metabolism
ribosomes
synthetic biology
systems biology

ASJC Scopus subject areas

Biomedical Engineering
Biochemistry, Genetics and Molecular Biology (miscellaneous)

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abstract = "Building variant ribosomes offers opportunities to reveal fundamental principles underlying ribosome biogenesis and to make ribosomes with altered properties. However, cell viability limits mutations that can be made to the ribosome. To address this limitation, the in vitro integrated synthesis, assembly and translation (iSAT) method for ribosome construction from the bottom up was recently developed. Unfortunately, iSAT is complex, costly, and laborious to researchers, partially due to the high cost of reaction buffer containing over 20 components. In this study, we develop iSAT in Escherichia coli BL21Rosetta2 cell lysates, a commonly used bacterial strain, with a cost-effective poly sugar and nucleotide monophosphate-based metabolic scheme. We achieved a 10-fold increase in protein yield over our base case with an evolutionary design of experiments approach, screening 490 reaction conditions to optimize the reaction buffer. The computationally guided, cell-free, high-throughput technology presented here augments the way we approach multicomponent synthetic biology projects and efforts to repurpose ribosomes.",

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AU - Jewett, Michael C.

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High-Throughput Optimization Cycle of a Cell-Free Ribosome Assembly and Protein Synthesis System

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