The genetic insulator RiboJ increases expression of insulated genes
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 phenom...
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doaj-6d760e7c564746518a70b9ced5d63af52020-11-25T01:37:19ZengBMCJournal of Biological Engineering1754-16112018-10-011211610.1186/s13036-018-0115-6The genetic insulator RiboJ increases expression of insulated genesKalen P. Clifton0Ethan M. Jones1Sudip Paudel2John P. Marken3Callan E. Monette4Andrew D. Halleran5Lidia Epp6Margaret S. Saha7Department of Biology, Integrated Science Center, The College of William and MaryDepartment of Biology, Integrated Science Center, The College of William and MaryDepartment of Biology, Integrated Science Center, The College of William and MaryDivision of Biology and Bioengineering, California Institute of TechnologyDepartment of Biology, Integrated Science Center, The College of William and MaryDivision of Biology and Bioengineering, California Institute of TechnologyDepartment of Biology, Integrated Science Center, The College of William and MaryDepartment of Biology, Integrated Science Center, The College of William and MaryAbstract 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.http://link.springer.com/article/10.1186/s13036-018-0115-6RiboJInsulationCharacterizationDigital droplet PCRGenetic circuitRibozyme |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Kalen P. Clifton Ethan M. Jones Sudip Paudel John P. Marken Callan E. Monette Andrew D. Halleran Lidia Epp Margaret S. Saha |
spellingShingle |
Kalen P. Clifton Ethan M. Jones Sudip Paudel John P. Marken Callan E. Monette Andrew D. Halleran Lidia Epp Margaret S. Saha The genetic insulator RiboJ increases expression of insulated genes Journal of Biological Engineering RiboJ Insulation Characterization Digital droplet PCR Genetic circuit Ribozyme |
author_facet |
Kalen P. Clifton Ethan M. Jones Sudip Paudel John P. Marken Callan E. Monette Andrew D. Halleran Lidia Epp Margaret S. Saha |
author_sort |
Kalen P. Clifton |
title |
The genetic insulator RiboJ increases expression of insulated genes |
title_short |
The genetic insulator RiboJ increases expression of insulated genes |
title_full |
The genetic insulator RiboJ increases expression of insulated genes |
title_fullStr |
The genetic insulator RiboJ increases expression of insulated genes |
title_full_unstemmed |
The genetic insulator RiboJ increases expression of insulated genes |
title_sort |
genetic insulator riboj increases expression of insulated genes |
publisher |
BMC |
series |
Journal of Biological Engineering |
issn |
1754-1611 |
publishDate |
2018-10-01 |
description |
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. |
topic |
RiboJ Insulation Characterization Digital droplet PCR Genetic circuit Ribozyme |
url |
http://link.springer.com/article/10.1186/s13036-018-0115-6 |
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