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.Electronic supplementary materialThe online version of this article (10.1186/s13036-018-0115-6) contains supplementary material, which is available to authorized users.

A fundamental principle of cellular signal processing is the encoding of information within the temporal dynamics of regulatory circuits. If synthetic circuits are to achieve the versatility and effectiveness of naturally-occurring circuits, it is necessary to develop simple, effective methods for the control of the dynamical properties of genetic circuits. However, current approaches to dynamical control often require extensive rewiring of circuit architecture, which hinders their implementation in a variety of systems. Therefore, it is essential that simple, modular, genetic parts-based frameworks are created to control the dynamical properties of circuits. Here we address this need by implementing a modular, genetic parts-based system which tunes the response time of a gene's expression by tuning its degradation rate via the application of protein degradation tags with various affinities to their protease. This system provides a simple, easilyapplicable framework for controlling the temporal aspects of genetic circuit behavior.

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The self-cleaving ribozyme RiboJ is an insulator commonly used in genetic circuits to prevent unexpected interactions between neighboring parts. These interactions can compromise the modularity of the circuit, impeding the implementation of predictable genetic constructs. Despite its utility as an insulator, a quantitative assessment of the effect of RiboJ on the properties of downstream genetic parts is lacking. 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. We show that depending on the strength of the promoters, insulation with RiboJ increased protein abundance between twofold and tenfold and increased transcript abundance by an average of twofold. This result is the first to demonstrate that genetic insulators can impact the expression of downstream genes, potentially hindering the design of predictable genetic circuits and constructs.