The lac repressor is a transcriptional regulator found in E.coli that monitors the levels of available lactose and adjusts expression of the genes involved in lactose utilization accordingly. Since its discovery, the lac operon has served as a model system for exploring the molecular mechanisms of gene regulation, protein-DNA recognition and allosteric signaling. In recent years, the molecular switch has become a valuable tool for regulating expression in a number of bacterial and eukaryotic systems. With such widespread application of the lac repressor, we saw the need and opportunity to design the next generation of transcriptional regulators. In this dissertation, we have used directed evolution to create repressors which improve upon the wild type system by reducing the leakiness of the switch. A series of novel repressors have also been developed which are capable of repressing non-classical operator sites. These repressors heterodimerize and allow two different DNA binding domains to function together in recognizing asymmetric operators. Finally, a series of repressors have been created that contain altered effector specificity. Several of these repressors induce with a previously neutral effector, ONPG, while other repressors function in the reverse direction by a mechanism of co-repression. In an effort to produce novel switches, we have also discovered facets of the lac operon that are responsible for optimal functionality. We demonstrate that the operator is not a passive component of the molecular switch: it is responsible for establishing binding affinity, specificity, and translational efficiency of the resulting transcript. Using the heterodimeric construct we were also able to determine that binding of two inducers is required for full induction. Finally, determination of crystal structures of the lac repressor bound to inducer and anti-inducer molecules provide a model for how these small molecules can modulate repressor function. These structures suggest that the O6 hydroxyl on the galactoside is essential for establishing a water-mediated hydrogen bonding network that bridges the N-terminal and C-terminal sub-domains. This hydrogen bonding can account in part for the different structural conformations of the repressor and is vital for the allosteric transition
