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.
Highlights
A fundamental goal of synthetic biology is the construction of genetic circuits that can perform a variety of useful functions that include environmental sensing and remediation, metabolic engineering, pharmaceutical and fuel production, drug delivery, and even cellular computation [1]
We found that insulation of constructs with RiboJ increased superfolder green fluorescent protein (sfGFP) transcript abundance by an average of twofold, while there was no change in transcript abundance of our reference gene Uroporphyrinogen-III C-methyltransferase (CysG) on average
The genetic insulator RiboJ is a valuable tool that aids the implementation of predictable genetic circuits by allowing promoter characterization to be standardized across genetic constructs
Summary
A fundamental goal of synthetic biology is the construction of genetic circuits that can perform a variety of useful functions that include environmental sensing and remediation, metabolic engineering, pharmaceutical and fuel production, drug delivery, and even cellular computation [1]. Similar to other engineering disciplines, a guiding tenet of circuit design is the use of well-characterized, modular parts—promoters, ribosome binding sites, coding regions—which in turn can be combined to produce a construct that behaves in a consistent, predictable manner [2]. This predictability relies on the use of parts whose behaviors are unaffected by other parts in the construct. One significant source of the effect of genetic context is the use of synthetic promoters containing regulatory sequences downstream of the transcriptional start site. Changes to a construct’s behavior will depend on both the specific promoter used and downstream composition of a construct (Fig. 1)
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