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The genetic insulator RiboJ increases expression of insulated genes

Clifton, Kalen P. and Jones, Ethan M. and Paudel, Sudip and Marken, John P. and Monette, Callan E. and Halleran, Andrew D. and Epp, Lidia and Saha, Margaret S. (2018) The genetic insulator RiboJ increases expression of insulated genes. Journal of Biological Engineering, 12 . Art. No. 23. ISSN 1754-1611. PMCID PMC6206723. https://resolver.caltech.edu/CaltechAUTHORS:20181029-094838044

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Abstract

A primary objective of synthetic biology is the construction of genetic circuits with behaviors that can be predicted based on the properties of the constituent genetic parts from which they are built. However a significant issue in the construction of synthetic genetic circuits is a phenomenon known as context dependence in which the behavior of a given part changes depending on the choice of adjacent or nearby parts. Interactions between parts compromise the modularity of the circuit, impeding the implementation of predictable genetic constructs. To address this issue, investigators have devised genetic insulators that prevent these unintended context-dependent interactions between neighboring parts. One of the most commonly used insulators in bacterial systems is the self-cleaving ribozyme RiboJ. Despite its utility as an insulator, there has been no systematic quantitative assessment of the effect of RiboJ on the expression level of downstream genetic parts. Here, we characterized the impact of insulation with RiboJ on expression of a reporter gene driven by a promoter from a library of 24 frequently employed constitutive promoters in an Escherichia coli model system. We show that, depending on the strength of the promoter, insulation with RiboJ increased protein abundance between twofold and tenfold and increased transcript abundance by an average of twofold. This result demonstrates that genetic insulators in E. coli can impact the expression of downstream genes, information that is essential for the design of predictable genetic circuits and constructs.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1186/s13036-018-0115-6DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6206723PubMed CentralArticle
https://doi.org/10.1101/317875DOIDiscussion Paper
ORCID:
AuthorORCID
Paudel, Sudip0000-0003-4502-8405
Marken, John P.0000-0001-9696-088X
Halleran, Andrew D.0000-0001-8720-1451
Saha, Margaret S.0000-0003-0096-2667
Additional Information:© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Received: 12 June 2018 Accepted: 11 October 2018. Published online: 29 October 2018. We would like to thank Joseph Maniaci for his assistance in the initial cloning of the construct library. We would also like to thank Vice Provost Dennis Manos for his intellectual support throughout the project. Research was funded by NSF Grant 1257895 to MSS and NIH Grant 1R15HD077624–01 to MSS. Project was also supported by funding from the office of the Vice Provost for Research and Graduate/Professional Studies at William & Mary. Kalen P. Clifton and Ethan M. Jones contributed equally to this work. Availability of data and materials: All data generated or analyzed during this study are included in this published article [and its Additional files 1 and 2]. Authors’ contributions: ADH designed constructs. EMJ and KPC cloned constructs with help of ADH. EMJ, KPC and SP isolated RNA. LE performed reverse transcription and digital droplet qPCR. EMJ and KPC performed flow cytometry measurements and analysis. EMJ, KPC and JPM analyzed data. KPC and JPM created figures. MSS supervised project. EMJ, KPC wrote manuscript. KPC, ADH EMJ, CEM, JPM, SP, MSS edited manuscript. All authors read and approved the final manuscript. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. The authors declare that they have no competing interests.
Funders:
Funding AgencyGrant Number
NSFIOS-1257895
NIH1R15HD077624-01
College of William and MaryUNSPECIFIED
Subject Keywords:RiboJ, Insulation, Characterization, Digital droplet PCR, Genetic circuit, Ribozyme
PubMed Central ID:PMC6206723
Record Number:CaltechAUTHORS:20181029-094838044
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20181029-094838044
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:90451
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:29 Oct 2018 18:09
Last Modified:09 Mar 2020 13:19

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