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Integrase-mediated differentiation circuits improve evolutionary stability of burdensome and toxic functions in E. coli

Williams, Rory L. and Murray, Richard M. (2022) Integrase-mediated differentiation circuits improve evolutionary stability of burdensome and toxic functions in E. coli. Nature Communications, 13 . Art. No. 6822. ISSN 2041-1723. PMCID PMC9649629. doi:10.1038/s41467-022-34361-y.

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Advances in synthetic biology, bioengineering, and computation allow us to rapidly and reliably program cells with increasingly complex and useful functions. However, because the functions we engineer cells to perform are typically burdensome to cell growth, they can be rapidly lost due to the processes of mutation and natural selection. Here, we show that a strategy of terminal differentiation improves the evolutionary stability of burdensome functions in a general manner by realizing a reproductive and metabolic division of labor. To implement this strategy, we develop a genetic differentiation circuit in Escherichia coli using unidirectional integrase-recombination. With terminal differentiation, differentiated cells uniquely express burdensome functions driven by the orthogonal T7 RNA polymerase, but their capacity to proliferate is limited to prevent the propagation of advantageous loss-of-function mutations that inevitably occur. We demonstrate computationally and experimentally that terminal differentiation increases duration and yield of high-burden expression and that its evolutionary stability can be improved with strategic redundancy. Further, we show this strategy can even be applied to toxic functions. Overall, this study provides an effective, generalizable approach for protecting burdensome engineered functions from evolutionary degradation.

Item Type:Article
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URLURL TypeDescription article CentralArticle ItemDiscussion Paper
Williams, Rory L.0000-0003-2605-5790
Murray, Richard M.0000-0002-5785-7481
Additional Information:The authors would like to thank the Sim and Liu Labs at the University of California Irvine for the use of lab space and equipment; Andrey Shur and Gordon Rix for help with MinION sequencing; Justin Bois, Andy Halleran, Anandh Swaminathan, and Andrey Shur for productive conversations; and Prof. Chang Liu, John Marken, and Gordon Rix for providing comments on the manuscript. NahRAM,, LasRAM, and LacIAM,, and their corresponding evolved promoters PSalTTC, PLasAM, and PTac were provided by Adam Meyer28. The CIDAR MoClo Parts Kit, which includes various promoter, RBS, CDS, and terminator parts used in the constructs described, was provided by Douglas Densmore (Addgene kit 1000000059). This research was supported by the Army Research Office (ARO) through grants W911NF-19-2-0026 and W911NF-09-D-0001.
Funding AgencyGrant Number
Army Research Office (ARO)W911NF-19-2-0026
Army Research Office (ARO)W911NF-09-D-0001
PubMed Central ID:PMC9649629
Record Number:CaltechAUTHORS:20221205-666301600.7
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ID Code:118234
Deposited By: Research Services Depository
Deposited On:06 Jan 2023 17:09
Last Modified:06 Jan 2023 17:39

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