A 'resource allocator' for transcription based on a highly fragmented T7 RNA polymerase
Name
Segall-Shapiro-2014-A resource allocator.pdf
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1.11 MB
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Author(s)
Segall-Shapiro, Thomas Hale
Meyer, Adam J.
Ellington, Andrew D.
Sontag, Eduardo D.
Voigt, Christopher A.
Date Issued
July 2014
Journal
Molecular Systems Biology
Publisher
EMBRO Press/Wiley
Citation
Segall-Shapiro, T. H., A. J. Meyer, A. D. Ellington, E. D. Sontag, and C. A. Voigt. “A ‘Resource Allocator’ for Transcription Based on a Highly Fragmented T7 RNA Polymerase.” Molecular Systems Biology 10, no. 7 (July 1, 2014): 742–742.
Version
Final published version
Abstract
Synthetic genetic systems share resources with the host, including machinery for transcription and translation. Phage RNA polymerases (RNAPs) decouple transcription from the host and generate high expression. However, they can exhibit toxicity and lack accessory proteins (σ factors and activators) that enable switching between different promoters and modulation of activity. Here, we show that T7 RNAP (883 amino acids) can be divided into four fragments that have to be co‐expressed to function. The DNA‐binding loop is encoded in a C‐terminal 285‐aa ‘σ fragment’, and fragments with different specificity can direct the remaining 601‐aa ‘core fragment’ to different promoters. Using these parts, we have built a resource allocator that sets the core fragment concentration, which is then shared by multiple σ fragments. Adjusting the concentration of the core fragment sets the maximum transcriptional capacity available to a synthetic system. Further, positive and negative regulation is implemented using a 67‐aa N‐terminal ‘α fragment’ and a null (inactivated) σ fragment, respectively. The α fragment can be fused to recombinant proteins to make promoters responsive to their levels. These parts provide a toolbox to allocate transcriptional resources via different schemes, which we demonstrate by building a system which adjusts promoter activity to compensate for the difference in copy number of two plasmids.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Synthetic Biology Center
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Creative Commons Attribution
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DOI of Published Version
https://doi.org/10.15252/msb.20145299
