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Hands-free control of heterologous gene expression in batch cultures

Olivier Borkowski, Drew Endy, Pakpoom Subsoontorn
doi: https://doi.org/10.1101/150375
Olivier Borkowski
aBioengineering Department, 443 Via Ortega Shriram Center, Room 235, Stanford University, Stanford, CA, 94305, USA
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Drew Endy
aBioengineering Department, 443 Via Ortega Shriram Center, Room 235, Stanford University, Stanford, CA, 94305, USA
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Pakpoom Subsoontorn
aBioengineering Department, 443 Via Ortega Shriram Center, Room 235, Stanford University, Stanford, CA, 94305, USA
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Abstract

Background Autonomous cell-based control of heterologous gene expression can simplify batch-culture bioprocessing by eliminating external monitoring and extrinsic control of culture conditions. Existing approaches use auto-induction media, synthetic cell-cell communication systems, or application-specific biosensors. A simpler, resource-efficient, and general-purpose expression control system responsive to common changes during batch culture would be useful.

Results We used native E.coli promoters and recombinase-based switches to repurpose endogenous transcription signals for control of heterologous gene expression. Specifically, natural changes in transcription from endogenous promoters result in recombinase expression at different phases of batch culture. So-expressed recombinases invert a constitutive promoter regulating expression of arbitrary heterologous genes. We realized reversible and single-use switching, reduced static and dynamic cell-to-cell variation, and overall expression amplification. We used “off-the-shelf” genetic parts and abstraction-based composition frameworks to realize reliable forward engineering of our synthetic genetic systems.

Conclusion We engineered autonomous control systems for regulating heterologous gene expression. Our system uses generic endogenous promoters to sense and control heterologous expression during growth-phase transitions. Our system does not require specialized auto-induction media, production or activation of quorum sensing, or the development of application-specific biosensors. Cells programmed to control themselves could simplify existing bioprocess operations and enable the development of more powerful synthetic genetic systems.

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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 June 16, 2017.
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Hands-free control of heterologous gene expression in batch cultures
Olivier Borkowski, Drew Endy, Pakpoom Subsoontorn
bioRxiv 150375; doi: https://doi.org/10.1101/150375
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Hands-free control of heterologous gene expression in batch cultures
Olivier Borkowski, Drew Endy, Pakpoom Subsoontorn
bioRxiv 150375; doi: https://doi.org/10.1101/150375

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