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Title Exploring the Sequence Landscape of the Four-helix Bundle Protein ROP using DeepSequencing
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Publication Date
Degree MS
Discipline/Department Biophysics
Degree Level masters
University/Publisher The Ohio State University
Abstract High throughput DNA sequencing technologies have revolutionized the field ofgenomics. It is now possible to generate huge amounts of sequencing data at significantlylower costs and greater speed. In this study, we have utilized Illumina deep sequencing toanalyze the fitness landscape of the four-helix bundle protein ROP. All possible singlepoint mutants of the 63 amino acid protein ROP were constructed. From the library ofsingle point mutants of ROP, variants were enriched 1,000 fold for activity by a growthselection for six rounds, which works based on ROP’s role in plasmid copy numberregulation. 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 wereindividually subjected to growth selection, and colony sequencing of random variantsfrom sixth round were mostly actives as expected based on prior knowledge of thesepositions. Colony sequencing of rounds zero (naives) and six from the actual libraryenrichment yielded promising results. Illumina deep sequencing was performed onrounds zero, three, six and eight. Paired-end reads were generated with each sequencecontaining short barcodes at each end to reveal the round and orientation. From the dataanalysis, relative enrichment for variants from different rounds lets us delve into thefitness landscape of ROP.
Subjects/Keywords Biophysics; Biochemistry; Deep sequencing; Illumina sequencing; ROP
Contributors Magliery, Thomas (Advisor)
Language en
Rights unrestricted ; This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.
Country of Publication us
Format application/pdf
Record ID oai:etd.ohiolink.edu:osu1376954254
Repository ohiolink
Date Indexed 2016-12-22
Grantor The Ohio State University

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