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A Method for Cost-Effective and Rapid Characterization of Engineered T7-based Transcription Factors by Cell-Free Protein Synthesis Reveals Insights into the Regulation of T7 RNA Polymerase-Driven Expression

John B. McManus, Richard M. Murray, Peter A. Emanuel, Matthew W. Lux
doi: https://doi.org/10.1101/614545
John B. McManus
aUS Army Combat Capabilities Development Command, Army Research Laboratory, 2800 Powder Mill Rd, Adelphi, MD 20783
bCalifornia Institute of Technology, Biology and Biological Engineering, 1200 East California Blvd, Pasadena, CA 91125
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Richard M. Murray
bCalifornia Institute of Technology, Biology and Biological Engineering, 1200 East California Blvd, Pasadena, CA 91125
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Peter A. Emanuel
CUS Army Combat Capabilities Development Command Chemical Biological Center, 8567 Ricketts Point Road, APG, MD 21010
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Matthew W. Lux
CUS Army Combat Capabilities Development Command Chemical Biological Center, 8567 Ricketts Point Road, APG, MD 21010
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  • For correspondence: matthew.w.lux.civ@mail.mil
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Abstract

The T7 bacteriophage RNA polymerase (T7 RNAP) serves as a model for understanding RNA synthesis, as a tool for protein expression, and as an actuator for synthetic gene circuit design in bacterial cells and cell-free extract. T7 RNAP is an attractive tool for orthogonal protein expression in bacteria owing to its compact single subunit structure and orthogonal promoter specificity. Understanding the mechanisms underlying T7 RNAP regulation is important to the design of engineered T7-based transcription factors, which can be used in gene circuit design. To explore regulatory mechanisms for T7 RNAP-driven expression, we developed a rapid and cost-effective method to characterize engineered T7-based transcription factors using cell-free protein synthesis and an acoustic liquid handler. Using this method, we investigated the effects of the tetracycline operator’s proximity to the T7 promoter on the regulation of T7 RNAP-driven expression. Our results reveal a mechanism for regulation that functions by interfering with the transition of T7 RNAP from initiation to elongation and validates the use of the method described here to engineer future T7-based transcription factors.

Highlights

  • Development of a rapid and cost-effective method for screening synthetic promoters.

  • Insights into the regulation of engineered T7-based transcription factors and T7 RNAP enzyme kinetics.

  • Validation of this method by comparison with the T7 RNAP kinetic model.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.
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Posted April 20, 2019.
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A Method for Cost-Effective and Rapid Characterization of Engineered T7-based Transcription Factors by Cell-Free Protein Synthesis Reveals Insights into the Regulation of T7 RNA Polymerase-Driven Expression
John B. McManus, Richard M. Murray, Peter A. Emanuel, Matthew W. Lux
bioRxiv 614545; doi: https://doi.org/10.1101/614545
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A Method for Cost-Effective and Rapid Characterization of Engineered T7-based Transcription Factors by Cell-Free Protein Synthesis Reveals Insights into the Regulation of T7 RNA Polymerase-Driven Expression
John B. McManus, Richard M. Murray, Peter A. Emanuel, Matthew W. Lux
bioRxiv 614545; doi: https://doi.org/10.1101/614545

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