Improving the Lac System for Synthetic Biology

The Escherichia coli lac Operon is controlled by a regulatory system that has been the subject of intensive study for the past fifty years. The system creates metabolic efficiency by responding to the levels of environmental lactose. In the absence of lactose, the LacI protein acts as a repressor of transcription from the lac promoter. Transcription begins when lactose binds to LacI, which results in the expression of three genes involved in lactose uptake and catabolism. The lac promoter is the most commonly used promoter in the field of synthetic biology. Although it is widely used, the lac promoter is known to have leaky transcription, meaning that transcription takes place even when the repressor is present and the inducer is absent. In an effort to redesign the lac promoter, we tested pLac variants that were reported to have a higher affinity for RNA polymerase than the wild-type. We also compared three mutants of the LacI repressor that were reported to have increasing affinity for the pLac promoter. Using GFP reporter constructs, we found that the pLacIQ1 promoter showed much higher levels of transcription than the wild-type promoter. Of the twelve combinations of promoters and repressors tested in the presence and absence of an inducer, we discovered that the wild-type LacI repressor protein with the pLacIQ1 mutant promoter is the best combination for high levels of induction and low levels of leaky transcription. Our results promise to help synthetic biologists design and build systems with tighter regulatory control.