UC Irvine

Natural evolution occurs when mutations to DNA confer an advantage to an organism facing a new or changing environment. However, this process is slow, occurring over millions of years. There is a great need in the chemicals and pharmaceutical industries among others, to be able to engineer proteins to catalyze specific reactions, bind new substrates, or function at different temperatures. Directed evolution is a method that enables protein engineering on laboratory timescales. However, traditional methods of directed evolution are only able to sample a limited number of protein variants during the course of the experiment and require significant user involvement at each stage, making them difficult to apply at large scales. The methods of continuous directed evolution, as described here, in particular OrthoRep, provide an ideal way to evolve proteins faster while simultaneously allowing the user to test multiple conditions and sample large sequence space, thus overcoming the limitations of traditional directed evolution methods. OrthoRep can be used to easily connect sequence with structure and function, an important step in understanding what mutations can lead to altered function. Here, I describe the advantages of OrthoRep, best practices to design experiments, and the molecular biology underpinning OrthoRep function in cells. To enable easy and widespread adoption of OrthoRep, I developed a modular toolkit and curated detailed protocols pertaining to its use and best practices. Additionally, there are examples of the kinds of experiments that may be performed and the kinds that are currently at the edge of what is possible using this system.