Directed evolution of AraC for improved compatibility of arabinose- and lactose-inducible promoters

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-beta-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.