Construction of AraC-based biosensors for target compounds in Escherichia coli using Multiplex Automated Genome Engineering

Abstract

AraC is an Escherichia coli regulatory protein which inhibits the expression of arabinose operon in the absence of inducer, arabinose, and activates the expression of the operon in the presence of arabinose. We describe a biosensor construction system with a PBAD-gfp reporter by engineering the Escherichia coli regulatory protein AraC to activate gene expression in response to target compounds. An araC library was constructed by simultaneous saturation mutagenesis in five amino acid positions located in the effector binding pocket (P8, T24, H80, Y82 and H93) using multiplex automated genome engineering(MAGE), randomly mutating those amino acid sites of AraC. MAGE can target many sites on the genome for mutation in a single cell, causing continuous directed evolution of cells. Thus, MAGE can give rise to large-scale genomic diversity. The library was screened for AraC variants responding to the presence of a target compound. This method can provide the utility of engineered AraC variants as customized molecular biosensors.