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Cooperative assembly confers regulatory specificity and long-term genetic circuit stability

Meghan D. J. Bragdon, Nikit Patel, James Chuang, Ethan Levien, Caleb J. Bashor, View ORCID ProfileAhmad S. Khalil
doi: https://doi.org/10.1101/2022.05.22.492993
Meghan D. J. Bragdon
1Biological Design Center, Boston University, Boston, MA 02215, USA
2Program in Molecular Biology, Cell Biology and Biochemistry, Boston University, Boston, MA 02215, USA
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Nikit Patel
1Biological Design Center, Boston University, Boston, MA 02215, USA
3Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA
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James Chuang
3Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA
4Department of Genetics, Harvard Medical School, Boston, MA 02115 USA
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Ethan Levien
5Department of Mathematics, Dartmouth College, Hanover, NH 03755 USA
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Caleb J. Bashor
6Department of Bioengineering, Rice University, Houston, TX 77030, USA
7Department of Biosciences, Rice University, Houston, TX 77030, USA
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Ahmad S. Khalil
1Biological Design Center, Boston University, Boston, MA 02215, USA
2Program in Molecular Biology, Cell Biology and Biochemistry, Boston University, Boston, MA 02215, USA
3Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA
8Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115 USA
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  • ORCID record for Ahmad S. Khalil
  • For correspondence: khalil@bu.edu
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ABSTRACT

In eukaryotes, links in gene regulatory networks are often maintained through cooperative self-assembly between transcriptional regulators (TRs) and DNA cis-regulatory motifs, a strategy widely thought to enable highly specific regulatory connections to be formed between otherwise weakly-interacting, low-specificity molecular components. Here, we directly test whether this regulatory strategy can be used to engineer regulatory specificity in synthetic gene circuits constructed in yeast. We show that circuits composed of artificial zinc-finger TRs can be effectively insulated from aberrant misregulation of the host cell genome by using cooperative multivalent TR assemblies to program circuit connections. As we demonstrate in experiments and mathematical models, assembly-mediated regulatory connections enable mitigation of circuit-driven fitness defects, resulting in genetic and functional stability of circuits in long-term continuous culture. Our naturally-inspired approach offers a simple, generalizable means for building evolutionarily robust gene circuits that can be scaled to a wide range of host organisms and applications.

Competing Interest Statement

N.P., C.J.B, and A.S.K. are co-inventors on a patent related to engineered cooperativity and control of gene expression. A.S.K. is a scientific advisor for and holds equity in Senti Biosciences and Chroma Medicine, and is a co-founder of Fynch Biosciences and K2 Biotechnologies.

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  • ↵10 Senior author

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted May 23, 2022.
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Cooperative assembly confers regulatory specificity and long-term genetic circuit stability
Meghan D. J. Bragdon, Nikit Patel, James Chuang, Ethan Levien, Caleb J. Bashor, Ahmad S. Khalil
bioRxiv 2022.05.22.492993; doi: https://doi.org/10.1101/2022.05.22.492993
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Cooperative assembly confers regulatory specificity and long-term genetic circuit stability
Meghan D. J. Bragdon, Nikit Patel, James Chuang, Ethan Levien, Caleb J. Bashor, Ahmad S. Khalil
bioRxiv 2022.05.22.492993; doi: https://doi.org/10.1101/2022.05.22.492993

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