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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. (2019) Integrase-mediated differentiation circuits improve evolutionary stability of burdensome and toxic functions in E. coli. . (Unpublished)

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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 unnecessary for cellular survival and burdensome to cell growth, they can be rapidly lost due to the processes of mutation and natural selection. To improve the evolutionary stability of engineered functions in a general manner, we developed an integrase-recombination-based differentiation gene circuit in Escherichia coli. In this system, differentiated cells uniquely carry out burdensome or toxic engineered functions but have limited capacity to grow (terminal differentiation), preventing the propagation of selectively advantageous loss of function mutations that inevitably arise. To experimentally implement terminal differentiation, we co-opted the R6K plasmid system, using differentiation to simultaneously activate T7 RNAP-driven expression of arbitrary engineered functions, and inactivate expression of π protein (an essential factor for R6K plasmid replication), thereby allowing limitation of differentiated cell growth through antibiotic selection. We experimentally demonstrate terminal differentiation increases both duration and magnitude of high-burden T7 RNAP-driven expression, and that its evolutionary stability can be further improved with strategic redundancy. Using burdensome overexpression of a fluorescent protein as a model engineered function, our terminal differentiation circuit results in a ~2.8-fold (single-cassette) and ~4.2-fold (two-cassette) increase of total fluorescent protein produced compared to high-burden naive expression in which all cells inducibly express T7 RNAP. Finally, we demonstrate that differentiation can enable the expression of even toxic functions, a feat not achieved to our knowledge by any other strategy for addressing long-term evolutionary stability. Overall, this study provides an effective generalizable strategy for protecting engineered functions from evolutionary degradation.

Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription Paper
Williams, Rory L.0000-0003-2605-5790
Murray, Richard M.0000-0002-5785-7481
Alternate Title:Tunable integrase-mediated differentiation facilitates improved output of burdensome functions in E. coli
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license. Version 1 - April 20, 2019; Version 2 - June 15, 2020; Version 3 - February 22, 2022; Version 4 - March 2, 2022. The authors would like to thank the Sim Lab at the University of California Irvine for use of lab space and equipment; Andrey Shur for his help with minION sequencing; 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. NahR^(AM), LasR^(AM), and LacI^(AM), and their corresponding evolved promoters P_(SalTTC), P_(LasAM), and P_(Tac) were provided by Adam Meyer.(27) The CIDAR MoClo Parts Kit which includes various promoter, RBS, CDS, and terminator parts used in the constructs described were provided by Douglas Densmore (Addgene kit 1000000059). This research was supported by the Army Research Office (ARO) through grant W911NF-19-2-0026, and by the Army Research Lab/Institute for Collaborative Biotechnologies (ARL/ICB) through grant W911NF-09-D-0001. The content of the information on this page does not necessarily reflect the position or the policy of the Government, and no official endorsement should be inferred. The authors declare that they have no conflict of interest.
Errata:During the course of continued efforts developing this integrase-mediated differentiation system for improving the evolutionary stability of burdensome engineered functions, we discovered that the E. coli strain with differentiation activated expression of T7 RNAP (Figure 4A) contains a second copy of the T7 RNAP differentiation cassette integrated into its genome. The T7 RNAP differentiation cassette was integrated into the genome using the clonetegration method with the phage 186 integrase, which has both primary and secondary landing sites on the E. coli genome.24 Though we verified the successful integration at the primary site with colony PCR and sequencing, we did not check the secondary site and discover the second integration until later experiments with improved versions of the differentiation system highlighted discrepancies in circuit behavior. Specifically, unpublished experiments indicate that the behavior of the T7 RNAP differentiation circuit in the experiments depicted in Figure 4E-F should show a larger performance difference between the (+)-chloramphenicol and (−)-chloramphenicol conditions. Revised versions of these experiments conducted with strains verified by whole genome sequencing will be included in an updated publication.
Funding AgencyGrant Number
Army Research Office (ARO)W911NF-19-2-0026
Army Research Office (ARO)W911NF-09-0001
Record Number:CaltechAUTHORS:20190422-091140053
Persistent URL:
Official Citation:Integrase-mediated differentiation circuits improve evolutionary stability of burdensome and toxic functions in E. coli. Rory. L Williams, Richard M. Murray. bioRxiv 614529; doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:94829
Deposited By: George Porter
Deposited On:22 Apr 2019 22:59
Last Modified:08 Mar 2022 19:41

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