System-level studies of a cell-free transcription-translation platform for metabolic engineering
Abstract
Current methods for assembling biosynthetic pathways in microorganisms require a process of repeated trial and error and have long design-build-test cycles. We describe the use of a cell-free transcription-translation (TX-TL) system as a biomolecular breadboard for the rapid engineering of the 1,4-butanediol (BDO) pathway. We demonstrate the reliability of TX-TL as a platform for engineering biological systems by undertaking a careful characterization of its transcription and translation capabilities and provide a detailed analysis of its metabolic output. Using TX-TL to survey the design space of the BDO pathway enables rapid tuning of pathway enzyme expression levels for improved product yield. Leveraging TX-TL to screen enzyme variants for improved catalytic activity accelerates design iterations that can be directly applied to in vivo strain development.
Additional Information
The copyright holder for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint first posted online Aug. 3, 2017. We thank Dr. Nathan Dalleska and the Environmental Analysis Center for the support and assistance using GC/MS for initial data collection. We thank Anna Lechner, Evan Ehrich, Emily Mitchell, Glenn Majer, and Sari Rizek for material analysis and preparation. We thank Jingyi Li, Jungik Choi, Jonathan Joaquin, Joseph R. Warner, Robin Osterhout, and Robert Haselbeck for helpful discussion. This material is based upon work supported in part by the Defense Advanced Research Projects Agency (DARPA/MTO) Living Foundries program; contract number HR0011-12-C-0065 (DARPA/CMO). Y.Y.W was supported by NIH/NRSA Training Grant 5T32 GM07616 and the Gordon and Betty Moore Foundation Grant GBMF2809 to the Caltech Programmable Molecular Technology Initiative.Attached Files
Submitted - 172007.full.pdf
Supplemental Material - 172007-1.pdf
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Additional details
- Eprint ID
- 86111
- Resolver ID
- CaltechAUTHORS:20180430-092750634
- HR0011-12-C-0065
- Defense Advanced Research Projects Agency (DARPA)
- 5T32 GM07616
- NIH Predoctoral Fellowship
- GBMF2809
- Gordon and Betty Moore Foundation
- Created
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2018-04-30Created from EPrint's datestamp field
- Updated
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2021-11-15Created from EPrint's last_modified field