Mechanistic Insights into Cell-Free Gene Expression through an Integrated -Omics Analysis of Extract Processing Methods.

Autor:	Rasor BJ; Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States.; Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois 60208, United States.; Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208, United States., Chirania P; Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States.; Graduate School of Genome Science and Technology, University of Tennessee, Knoxville, Tennessee 37996, United States., Rybnicky GA; Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois 60208, United States.; Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208, United States.; Interdisciplinary Biological Sciences Graduate Program, Northwestern University, Evanston, Illinois 60208, United States., Giannone RJ; Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States., Engle NL; Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States., Tschaplinski TJ; Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States., Karim AS; Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States.; Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois 60208, United States.; Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208, United States., Hettich RL; Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States., Jewett MC; Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States.; Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois 60208, United States.; Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208, United States.; Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, Illinois 60611, United States.; Simpson Querrey Institute, Northwestern University, Chicago, Illinois 60611, United States.
Jazyk:	angličtina
Zdroj:	ACS synthetic biology [ACS Synth Biol] 2023 Feb 17; Vol. 12 (2), pp. 405-418. Date of Electronic Publication: 2023 Jan 26.
DOI:	10.1021/acssynbio.2c00339
Abstrakt:	Cell-free systems derived from crude cell extracts have developed into tools for gene expression, with applications in prototyping, biosensing, and protein production. Key to the development of these systems is optimization of cell extract preparation methods. However, the applied nature of these optimizations often limits investigation into the complex nature of the extracts themselves, which contain thousands of proteins and reaction networks with hundreds of metabolites. Here, we sought to uncover the black box of proteins and metabolites in Escherichia coli cell-free reactions based on different extract preparation methods. We assess changes in transcription and translation activity from σ 70 promoters in extracts prepared with acetate or glutamate buffer and the common post-lysis processing steps of a runoff incubation and dialysis. We then utilize proteomic and metabolomic analyses to uncover potential mechanisms behind these changes in gene expression, highlighting the impact of cold shock-like proteins and the role of buffer composition.
Databáze:	MEDLINE
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