Exploring the Sequence Landscape of the Four-helix Bundle Protein ROP using DeepSequencing

Abstract

High throughput DNA sequencing technologies have revolutionized the field of genomics.It is now possible to generate huge amounts of sequencing data at significantly lower costs and greater speed.In this study, we have utilized Illumina deep sequencing to analyze the fitness landscape of the four-helix bundle protein ROP.All possible single point mutants of the 63 amino acid protein ROP were constructed.From the library of single point mutants of ROP, variants were enriched 1,000 fold for activity by a growth selection for six rounds, which works based on ROP's role in plasmid copy number regulation.The enrichment was monitored by a cell-based screen using a GFP reporter.As a proof of principle experiment, point mutant libraries at positions 14, 30 and 43 were individually subjected to growth selection, and colony sequencing of random variants from sixth round were mostly actives as expected based on prior knowledge of these positions.Colony sequencing of rounds zero (naives) and six from the actual library enrichment yielded promising results.Illumina deep sequencing was performed on rounds zero, three, six and eight.Paired-end reads were generated with each sequence containing short barcodes at each end to reveal the round and orientation.From the data analysis, relative enrichment for variants from different rounds lets us delve into the fitness landscape of ROP.iii Dedication To my mom, dad and family