Abstract
In the directed evolution of enzymes, the starting protein template used is a critical factor in determining the rate at which desirable mutations accumulate. Therefore, a more “evolvable” template capable of supporting a wider range of positive and neutral mutations will lead to faster and more efficient evolution. Ancestral proteins resurrected through ancestral sequence reconstruction (ASR) have been observed to act as highly stable generalists with broader functionalities than modern proteins, and as a result, they are believed to harbor more evolutionary potential than their modern homologs. Using ancestral and modern mutant libraries of the essential enzyme adenylate kinase (ADK) in E. coli with genomic ADK knocked out, evolvability can be assessed and compared through selection experiments performed under the selective pressure of low temperature. Establishment of these cell lines has proven challenging, particularly with regards to persistence of the plasmid pKoComp, which keeps cells alive between knockout of genomic ADK and transformation of library ADK but must later be removed for selection to take place. Additional challenges have arisen regarding the performance and reliability of our custom turbidostat bioreactor, which automates the process of diluting cell cultures to a desirable cell density during selection. In order to streamline and improve the selection process, modifications to the turbidostat bioreactor have improved the instrument’s performance and reliability, and the design and implementation of a conditional toxin counterselection system has shown promise as an additional means of ensuring pKoComp removal.