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Published September 2013 | Published + Supplemental Material
Journal Article Open

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Abstract

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform. This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology. To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems. The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted. The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters. Accompanying mathematical models are available. The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Additional Information

© 2013 Journal of Visualized Experiments. Date Published: 9/16/2013. We thank Jongmin Kim, Dan Siegal-Gaskins, Anu Thubagere, and Enoch Yeung for assistance streamlining the protocol, and Clare Chen and Barclay Lee for assistance in the early stages of the project. This material is based upon work supported in part by the Defense Advanced Research Projects Agency (DARPA/MTO) Living Foundries program, contract number HR0011-12-C-0065 (DARPA/CMO.Z.Z.S. is also supported by a UCLA/Caltech Medical Scientist Training Program fellowship and by a DoD, Air Force Office of Scientific Research, National Defense Science and Engineering Graduate (NDSEG) Fellowship, 32 CFR 168a. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing officially policies, either expressly or implied, of the Defense Advanced Research Projects Agency or the U.S. Government.

Attached Files

Published - jove-protocol-50762-protocols-for-implementing-an-escherichia-coli-based-tx-tl-cell-free.pdf

Supplemental Material - TXTL_JoVE.xlsx

Supplemental Material - TXTL_e_template__calibration_JoVE.xlsx

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Created:
August 19, 2023
Modified:
October 25, 2023