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Title: De novo design of programmable inducible promoters

Abstract

Abstract Ligand-responsive allosteric transcription factors (aTF) play a vital role in genetic circuits and high-throughput screening because they transduce biochemical signals into gene expression changes. Programmable control of gene expression from aTF-regulated promoter is important because different downstream effector genes function optimally at different expression levels. However, tuning gene expression of native promoters is difficult due to complex layers of homeostatic regulation encoded within them. We engineered synthetic promoters de novo by embedding operator sites with varying affinities and radically reshaped binding preferences within a minimal, constitutive Escherichia coli promoter. Multiplexed cell-based screening of promoters for three TetR-like aTFs generated with this approach gave rich diversity of gene expression levels, dynamic ranges and ligand sensitivities and were 50- to 100-fold more active over their respective native promoters. Machine learning on our dataset revealed that relative position of the core motif and bases flanking the core motif play an important role in modulating induction response. Our generalized approach yields customizable and programmable aTF-regulated promoters for engineering cellular pathways and enables the discovery of new small molecule biosensors.

Authors:
 [1];  [2];  [3];  [2]; ORCiD logo [4];  [5]; ORCiD logo [6]
  1. Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA, The Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53706, USA
  2. The Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53706, USA, Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
  3. Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA
  4. Department of Biology, University of Puerto Rico-Rio Piedras, San Juan, PR 00925, USA
  5. Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA, The Genome Center of Wisconsin, University of Wisconsin-Madison, Madison, WI 53706, USA
  6. Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA, The Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53706, USA, The Genome Center of Wisconsin, University of Wisconsin-Madison, Madison, WI 53706, USA, Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA
Publication Date:
Sponsoring Org.:
USDOE
OSTI Identifier:
1566242
Grant/Contract Number:  
SC0018409; FC02-07ER64494
Resource Type:
Published Article
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Name: Nucleic Acids Research Journal Volume: 47 Journal Issue: 19; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Country of Publication:
United Kingdom
Language:
English

Citation Formats

Liu, Xiangyang, Gupta, Sanjan T. P., Bhimsaria, Devesh, Reed, Jennifer L., Rodríguez-Martínez, José A., Ansari, Aseem Z., and Raman, Srivatsan. De novo design of programmable inducible promoters. United Kingdom: N. p., 2019. Web. doi:10.1093/nar/gkz772.
Liu, Xiangyang, Gupta, Sanjan T. P., Bhimsaria, Devesh, Reed, Jennifer L., Rodríguez-Martínez, José A., Ansari, Aseem Z., & Raman, Srivatsan. De novo design of programmable inducible promoters. United Kingdom. doi:10.1093/nar/gkz772.
Liu, Xiangyang, Gupta, Sanjan T. P., Bhimsaria, Devesh, Reed, Jennifer L., Rodríguez-Martínez, José A., Ansari, Aseem Z., and Raman, Srivatsan. Wed . "De novo design of programmable inducible promoters". United Kingdom. doi:10.1093/nar/gkz772.
@article{osti_1566242,
title = {De novo design of programmable inducible promoters},
author = {Liu, Xiangyang and Gupta, Sanjan T. P. and Bhimsaria, Devesh and Reed, Jennifer L. and Rodríguez-Martínez, José A. and Ansari, Aseem Z. and Raman, Srivatsan},
abstractNote = {Abstract Ligand-responsive allosteric transcription factors (aTF) play a vital role in genetic circuits and high-throughput screening because they transduce biochemical signals into gene expression changes. Programmable control of gene expression from aTF-regulated promoter is important because different downstream effector genes function optimally at different expression levels. However, tuning gene expression of native promoters is difficult due to complex layers of homeostatic regulation encoded within them. We engineered synthetic promoters de novo by embedding operator sites with varying affinities and radically reshaped binding preferences within a minimal, constitutive Escherichia coli promoter. Multiplexed cell-based screening of promoters for three TetR-like aTFs generated with this approach gave rich diversity of gene expression levels, dynamic ranges and ligand sensitivities and were 50- to 100-fold more active over their respective native promoters. Machine learning on our dataset revealed that relative position of the core motif and bases flanking the core motif play an important role in modulating induction response. Our generalized approach yields customizable and programmable aTF-regulated promoters for engineering cellular pathways and enables the discovery of new small molecule biosensors.},
doi = {10.1093/nar/gkz772},
journal = {Nucleic Acids Research},
number = 19,
volume = 47,
place = {United Kingdom},
year = {2019},
month = {9}
}

Journal Article:
Free Publicly Available Full Text
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DOI: 10.1093/nar/gkz772

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