Graded Expression of A Protein Under Positive Selection

Source: Neuenschwander et al. (2008). A simple selection strategy for evolving highly efficient enzymes. Nat Biotech 25 (10): 1145-47.

This paper discusses a more robust method for directed evolution. Many enzymes can be subjected to evolution if they can be engineered into a background where their activity is essential for growth and survival of the host. This approach works find but there are a number of obstacles. For example, growth is a broad phenotype; even a limited activity of the enzyme might be sufficient to sustain growth resulting in the saturation of the evolution process. In this paper, he authors make the case that by a gradual decrease in the transcription, translation and half-life of the protein of interest, we can reduce the concentration of the available enzyme thus pushing it back into the optimization process. As shown below, their experimental set-up includes an inducible tet promoter and an ssrA for protein degradation and lowerig half life.