Fourteen Ways to Reroute Cooperative Communication in the Lactose Repressor: Engineering Regulatory Proteins with Alternate Repressive Functions

The lactose repressor (Lad) is a classic genetic switch that has been used as a fundamental component in a host of synthetic genetic networks. To expand the function of Lad for use in the development of novel networks and other biotechnological applications, we engineered alternate communication in the Lacl scaffold via laboratory evolution. Here we produced 14 new regulatory elements based on the Lad topology that are responsive to isopropyl beta-D-1-thiogalactopyranoside (IPTG) with variation in repression strengths and ligand sensitivities-on solid media. The new variants exhibit repressive as well as antilac (i.e., inverse-repression + IPTG) functions and variations in the control of gene output upon exposure to different concentrations of IPTG. In addition, examination of this collection of variants in solution results in the controlled output of a canonical florescent reporter, demonstrating the utility of this collection of new regulatory proteins under standard conditions.