An Experimental and Computational Evolution-Based Method to Study a Mode of Co-evolution of Overlapping Open Reading Frames in the AAV2 Viral Genome
Figure 2
The approach used for the experimental directed evolution of the VP/AAP-overlapping ORFs.
(A) Map of VP3 and AAP mutant plasmid libraries. Red bars indicate the random heptapeptide region corresponding to the QVKEVTQ/KSKRSRR motifs. (B) Schematic representation of the AAV directed evolution procedure. The numbers 1 through 5 in the figure indicate steps in the procedure. Step 1, Construction of the original plasmid DNA library (pAAV2-Lib-0); Step 2, Production of AAV virus library (AAV2-Lib-1); Step 3, Recovery of viral genomic DNA; Step 4, PCR amplification of the heptapeptide-coding region in the viral genome; Step 5, Construction of the next round plasmid DNA library using the PCR amplicons from Step 4 (pAAV2-Lib-1). Then Step 2 follows for the next round positive selection. This procedure was repeated three times to obtain AAV2-Lib-2 and AAV2-Lib-3. (C) The timeline of Illumina sequencing of PCR amplicons of the heptapeptide region. Illumina sequencing was used for extensively characterize the heptapeptides evolved by the experimental procedure.