Designing a genetic toggle switch for E. coli that uses sequestration of a eukaryotic repressor as a mechanism for ultrasensitivity

Abstract

Bistable gene expression—when a gene’s output can achieve and alternate between two distinct, stable states—plays a critical role in the regulation of various cell and developmental processes including cell-cycle progression, differentiation, and signaling. To study and harness this regulatory process in bacteria, synthetic biologists have created gene circuits in E. coli that use sequestration of bacterial activators driving their own expression by inducible inhibitors to generate ultrasensitive positive feedback that leads to bistability in their expression. While capable of bistability, these circuits can be affected by cross-interference with native E. coli regulatory processes and cause toxic squelching that make studying these circuits difficult. As such, gene circuits that can produce bistable gene expression in E. coli via sequestration-based ultrasensitivity while avoiding cross-interference and toxic squelching would be valuable tools for synthetic biology. Based on a premise that using eukaryotic repressors should avoid both toxic squelching and cross-interference in bacterial hosts, I here present efforts to create a circuit in E. coli that uses sequestration of the eukaryotic repressor C/EBP by a synthetic inhibitor called 3HF to generate bistable gene expression. While I did not obtain a working circuit, I made progress toward selecting promoters and replication origins that balance the expressions of C/EBP and 3HF, and toward selecting a fluorescent protein tag that is compatible with 3HF in E. coli.