The Genetic Insulator RiboJ Increases Expression of Insulated Genes

Abstract

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.