Directed Evolution of AraC for Improved Compatibility of Arabinose- and Lactose-Inducible Promoters

ABSTRACT Synthetic biological systems often require multiple, independently inducible promoters in order to control the expression levels of several genes; however, cross talk between the promoters limits this ability. Here, we demonstrate the directed evolution of AraC to construct an arabinose-inducible (P BAD ) system that is more compatible with IPTG (isopropyl-β- d -1-thiogalactopyranoside) induction of a lactose-inducible (P lac ) system. The constructed system is 10 times more sensitive to arabinose and tolerates IPTG significantly better than the wild type. Detailed studies indicate that the AraC dimerization domain and C terminus are important for the increased sensitivity of AraC to arabinose.