DNA Component Order Independence Using Insulated Bacterial Promoters

Unexpected impact from flanking DNA sequences has been identified as an important cause of failed a priori genetic circuit design. Genetic parts characterized in one context often experience unexpected changes in expression level when moved into the context of their target genetic device. Furthermore, individual transcriptional units (TUs) or modules expected to be orthogonal to others within a device are, in practice, dependent upon the order in which they are arranged within a synthetic genetic device.  Effective insulator techniques have been developed for the promoter::RBS and RBS::gene boundaries, but no systematic method for insulating promoters from their upstream context has previously proven effective.

We address the issue of upstream promoter context dependence with a novel method based on a randomized insulator library: we have developed a high-throughput, flow cytometry-based screen that randomly samples from a library of 4³⁶ insulators created in a single cloning step. Screened against a reference device, our method provides precise control over genetic circuit expression. This new method of custom-insulator selection can be performed on both constitutive and transcriptionally regulated bacterial promoters, and can also be used to tune promoter expression level to a desired dynamic range. Finally, we validate the efficacy of this method for insulating promoters by showing that insulating the promoters in a genetic NOT-gate circuit using this method improves circuit performance and nearly eliminates the effect of the order in which the promoters are organized in the device.