(1990) Mutations affecting primer RNA interaction with the replication repressor RNA I in plasmid CoIE1: potential RNA folding pathway mutants. (1990) Roles of phi X174 type primosome- and G4 type primase-dependent primings in initiation of lagging and leading strand syntheses of DNA replication. (2010) Modulation of ColE1-like plasmid replication for recombinant gene expression. (1991) Control of ColE1 plasmid replication by antisense RNA. (1988) ColE1 replication control circuitry: sense from antisense. Here, we present examples illustrating these observations as well as our methods for efficient quantification of plasmid copy number. Thus, plasmid dosage may contribute significantly to evolution by fine-tuning levels of activity. The selected plasmid ori mutations are diverse and produce a range of plasmid copy numbers, suggesting a complex interplay between ori and coding mutations rather than a simple enhancement of level of expression of the target gene. Surprisingly, this is true even during evolution of new biochemical activities, when the activity that is being selected was not originally present. We have observed that when random mutations are not restricted to the coding sequence of the target genes, directed evolution results in a strong positive selection of plasmid origin of replication ( ori) mutations. Our target genes are encoded in ColE1 plasmids to facilitate the generation of libraries in vivo. Our laboratory specializes in directed protein evolution, i.e., evolution of proteins under defined selective pressures in the laboratory.
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