A load driver device for engineering modularity in biological networks

Abstract

The behavior of gene modules in complex synthetic circuits is often unpredictable1–4. Upon joining modules to create a circuit, downstream elements (such as binding sites for a regulatory protein) apply a load to upstream modules that can negatively affect circuit function1,5. Here we devise a genetic device named a load driver that mitigates the impact of load on circuit function, and we demonstrate its behavior in Saccharomyces cerevisiae. The load driver implements the design principle of time scale separation: inclusion of the load driver’s fast phosphotransfer processes restores the capability of a slower transcriptional circuit to respond to time-varying input signals even in the presence of substantial load. Without the load driver, we observe circuit behavior that suffers from 76% delay in response time and a 25% decrease in system bandwidth due to load. With the addition of a load driver, circuit performance is almost completely restored. Load drivers will serve as fundamental building blocks in the creation of complex, higher level genetic circuits.

A Synthetic Mammalian Therapeutic Gene Circuit for Sensing and Suppressing Inflammation

“…On the other hand, when designing complex gene circuits by joining several modules, decoy DNA binding sites can apply a load to a system and negatively influence circuit function, which was alleviated by a designed load driver device. 49 However in this study, we did not test how the thresholder affected ultra-sensitivity of the system, because our primary goal was to decrease the transcriptional leakage of the circuit. Upon introduction of the thresholder, our circuit retained graded response, which depended on the concentration of the input inflammatory signals ( Figure 4A).…”

An N-stage cascade of phosphorylation cycles as an insulation device for synthetic biological circuits

“…1b. This loading phenomenon has been experimentally shown both in vivo and in vitro in bacteria and yeast [12], [7], [10].…”

“…This tradeoff was theoretically characterized in [9] and experimentally verified using a NRI-NRI * PD cycle [8]. The results of [10] suggest that this tradeoff may be overcome by using multiple stages of PD cycles. In [11], a cascade of PD cycles are analyzed for the propogation of downstream disturbances to the input, and sufficient conditions for attenuating these disturbances are provided.…”

Modularity, context-dependence, and insulation in engineered biological circuits

“…Based on this principle, if the upstream and downstream systems are genetic circuits, with characteristic slow time scales dictated by gene expression, an intervening insulation device can be designed employing a number of processes, including phosphorylation, phosphotransfer, methylation, or their combinations, since they have relatively faster time scales. A particular instance of an insulation device, called the load driver, was constructed based on this principle in yeast cells employing a two-stage YPD1/SKN7 phosphotransfer cascade [43]. This device has a remarkable ability of attenuating retroactivity, applies no substantial load to its upstream system, and has an extremely fast response, therefore satisfying all three requirements of an insulation device.…”