Construction and Engineering of Positive Feedback Loops

Artificial positive feedback loops (PFLs) have been used as genetic amplifiers for enhancing the responses of weak promoters and in the creation of eukaryotic gene switches. Here we describe the construction and directed evolution of two PFLs based on the LuxR transcriptional activator and its cognate promoter, P luxI . The wild-type PFLs are completely activated by 10 nM of 3-oxo-hexanoyl-homoserine lactone (OHHL). Directed evolution of LuxR increased the sensitivity of the feedback loops, resulting in systems that are completely activated at OHHL concentrations of 5 nM, or ~3 molecules per cell. The responses of the PFLs can also be modulated by adjusting inducer concentrations. These highly sensitive yet regulatable PFLs can be used to construct larger artificial genetic networks to gain understanding of the design principles of complex biological systems and are expected to find various applications in industrial fermentation and gene therapy.