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Tunable integrase-mediated differentiation facilitates improved output of burdensome functions in E. coli

Williams, Rory L. and Murray, Richard M. (2019) Tunable integrase-mediated differentiation facilitates improved output of burdensome functions in E. coli. . (Unpublished)

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Application of synthetic biology is limited by the capacity of cells to faithfully execute burdensome engineered functions in the face of Darwinian evolution. Division of labor, both metabolic and reproductive, are underutilized in confronting this barrier. To address this, we developed a serine-integrase based differentiation circuit that allows control of the population composition through tuning of the differentiation rate and number of cell divisions differentiated cells can undergo. We applied this system to T7 RNAP-driven expression of a fluorescent protein, and demonstrate both increased duration of circuit function and total production for high burden expression. While T7 expression systems are typically used for high-level short-term expression, this system enables longer duration production, and could be readily applied to burdensome or toxic products not readily produced in bacteria.

Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription Paper Information
Williams, Rory L.0000-0003-2605-5790
Murray, Richard M.0000-0002-5785-7481
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. We would like to thank Andy Halleran, Anandh Swaminathan, and Andrey Shur for productive conversations; Andy Halleran for providing code for analysis of flow cytometry data; Samuel Clamons for providing code for tidying and analyzing Biotek data; and Andrey Shur and Andy Halleran for providing cloning resources. pSal, pLas, and pTac and their associated evolved transcription factors were kind gifts from Adam Meyer. The CIDAR MoClo Parts Kit was a gift from Douglas Densmore (Addgene kit # 1000000059). This research is supported by the Institute for Collaborative Biotechnologies through grant W911NF-09-0001 and cooperative agreement W911NF-19-2-0026 from the U.S. Army Research Office. 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.
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-09-0001
Army Research Office (ARO)W911NF-19-2-0026
Record Number:CaltechAUTHORS:20190422-091140053
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Official Citation:Tunable integrase-mediated differentiation facilitates improved output of burdensome 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:16 Nov 2021 17:08

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