(530e) Generating Libraries for Directed Evolution of Proteins – Comparison of Recombination-Dependant Pcr and DNA Shuffling

Authors: 
Loo, B., GIT
Powers, A., GIT
Bommarius, A. S., Georgia Institute of Technology


Good library creation is the most important step in successful directed evolution experiments. The screening of suboptimal libraries that contain a high background of parental templates is a waste of time and resources.

In this investigation, we have compared systematically the utility of DNA-shuffling protocols with recombination-based PCR protocols on b-lactamase and fluorescent proteins. DNA-shuffling is a commonly used method to generate libraries, but is disadvantaged by the presence of a high parental background. We used variations of recombination-based PCR protocols which necessitates at least 1 crossover for amplification.

Working towards the goal of evaluating the recombination protocols for different recombination needs, we created 3 scenarios of gene recombination. In the first case, recombination of point mutations were done on b-lactamase gene using DNA-shuffling and recombination-based PCR protocols. In the second scenario, we recombined highly homologous genes of >70% from fluorescent proteins. Lastly, we explored the limits of the lowest homology we can recombine with the current recombination protocols. The findings provide a guide for more efficient and successful gene recombination.