Design and Screening of Degenerate-Codon-Based Protein Ensembles with M13 Bacteriophage by Griffin James Clausen B.S. Biomedical Engineering University of Minnesota – Twin Cities, 2012 Submitted to the Department of Biological Engineering in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biological Engineering at the Massachusetts Institute of Technology February 2019 © 2019 Massachusetts Institute of Technology. All rights reserved. Signature of Author: ____________________________________________________________ Griffin Clausen Department of Biological Engineering January 14, 2019 Certified by: ___________________________________________________________________ Angela Belcher James Mason Crafts Professor of Biological Engineering and Materials Science Thesis Supervisor Accepted by: ___________________________________________________________________ Forest White Professor of Biological Engineering Graduate Program Committee Chair This doctoral thesis has been examined by the following committee: Amy Keating Thesis Committee Chair Professor of Biology and Biological Engineering Massachusetts Institute of Technology Angela Belcher Thesis Supervisor James Mason Crafts Professor of Biological Engineering and Materials Science Massachusetts Institute of Technology Paul Blainey Core Member, Broad Institute Associate Professor of Biological Engineering Massachusetts Institute of Technology 2 Design and Screening of Degenerate-Codon-Based Protein Ensembles with M13 Bacteriophage by Griffin James Clausen Submitted to the Department of Biological Engineering on January 14, 2019 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biological Engineering Abstract A billion years of evolution has crafted a diverse set of proteins capable of complex and varied functionalities. Within recent decades, scientists have applied both rational design and directed evolution to accelerate development of high-value proteins, including biotherapeutics. While computational modelling increasingly facilitates protein design, empirically screening large collections of protein variants remains an essential component of protein engineering. This process requires generating protein variation, partitioning variants with a selection pressure, and identifying highly functional proteins. This thesis presents computational tools for initial protein library design, leverages high-throughput sequencing for phage display screenings, and reports biotemplating of an inorganic phase-change material onto the filamentous M13 phage surface. Designing ensembles of protein variants involves optimizing library size and quality with constraints on screening capacity, cost, and experimental complexity. Incorporating degenerate codons during oligonucleotide synthesis enables residue-specific protein randomization with a known amino acid scope. However, this widely adopted method often generates uneven variant abundances that diverge exponentially with additional randomized residues. The first section of this work presents tools for the design and assessment of degenerate-codon-based protein libraries. This framework facilitates incorporating an arbitrarily large number of randomized sites, non-standard genetic codes, and non-equimolar nucleotide mixtures. In addition to library size and coverage calculations, whole-population diversity metrics and abundance quantiles are reported. An evolutionary solver to optimize non-equimolar base compositions to match amino acid profiles, as well as mutational profiling for spike-in oligonucleotides is also presented. The second section of this thesis develops an experimental and data analysis pipeline for integrating high-throughput DNA sequencing with M13 phage display biopanning. Deeply sequencing naïve M13 peptide libraries elucidated censorship patterns for both p3 and p8 coat protein fusions. Streptavidin panning recapitulated the HPQ binding motif after a single panning round, and additional biopannings pursued M13 p8 variants that interact with both gold films and carbon nanotubes. Furthermore, this thesis explores the effect of M13 p8 surface charge on the biotemplating of an inorganic phase-change material. An ambient temperature synthesis for modulating the atomic composition of germanium-tin-oxide nanomaterials is reported. Thesis Supervisor: Prof. Angela Belcher Title: James Mason Crafts Professor of Biological Engineering and Materials Science 3 Acknowledgements First and foremost, I would like to thank my advisor, Angela Belcher, for her guidance throughout my graduate experience. Angie brings a contagious enthusiasm and creativity to science that can inspire schoolchildren, graduate students, and grey-haired alumni alike. I was drawn to the breadth of research and the ambitious applications pursued by the Belcher Lab, and have learned a tremendous amount over the last few years in this unique environment. Furthermore, I would like to thank my thesis committee members, Amy Keating and Paul Blainey for providing valuable feedback on my graduate research and guidance for my future career. I am very grateful to the members of the Belcher lab for the friendship and assistance generously shared with me. Nimrod, thank you for our countless hours of phage-centric discussion and your second-opinions on perplexing plates. Thank you, Gaelen and Rana, for helping me settle into the lab and launching the deep sequencing project. Thank you to the Bio Subgroup for the illuminating discussions and thank you, Uyanga, for organizing these valuable sessions. Thank you, Eric, for your leadership and willingness to tackle tasks that aim to benefit the broader lab. Thank you, Ngozi, for being a talented community-builder, researcher, reviewer, and ice-cream- cake-shopper. Peter, thank you for the endless conversations. Thank you, Jifa, for your assistance during my 20.109 TA-ship. Thank you, John Casey, for showing me the EHS Rep ropes and thank you, Archana, for taking on the EHS Rep ropes. Thank you, Jackie, Matt, Yu, Nurxat, Xiangnan, Neel, Alan, Will, Swati, George, Briana, Shalmalee, and everyone else! Through my research, I had the pleasure of working with two incredibly hard-working collaborators. Thank you, Professor Desmond Loke, for leading the germanium tin oxide nanowire project as well as our Vermont hiking expedition. Thank you, Dr. Anthony Rojas from the Pentelute Lab, for the expertise and reagents you developed for our exploration of cysteine bioconjugation on phage. I was also fortunate enough to have the assistance of several bright UROPS: Jelle van der Hilst, Devany West, and Pearl Lee. Each of whom made valuable contributions towards the mutagenesis and cloning of phage, peptide conjugation, biopanning experiments, and binding validation assays. While EHS Representative for the lab, I had the chance to engage with many diligent MIT Environment, Health, & Safety employees. Thank you for your guidance Jennifer Lynn, Hans Richter, Wilfred Mbah, Carolyn Stahl, and Roberta Polak. To my fellow Biological Engineering cohort – thank you! Grinding through implementations and exams, with memorable celebrations in Puerto Rico; ski trips and broken wrists; softball and wings; Olympic victories and talent shows; you have left me with many wonderful memories and lasting friendships. And to my excellent 9 Seattle housemates: Santi, Shawn, Kelly, Truby, Frances, Claire, and Khoi- you are such an impressive and fun group of friends that have made my Boston experience unforgettable. Finally, I would like to thank my family. To my parents- thank you for the constant love and support. To my Grandma, thank you for the stream of letters and thoughts. And to Ali, thank you for the endless encouragement and shared excitement for the future. 4 Design and Screening of Degenerate-Codon-Based Protein Ensembles with M13 Bacteriophage Chapters 1 Introduction to M13 Bacteriophage, Protein Libraries, & Ligand Screening 2 Abundance Disparity and Sampling Coverage in Large Degenerate-Oligonucleotide- Based Protein Library Designs 3 High-Throughput Sequencing of Phage-Displayed Libraries and Biopanning Against Biological and Inorganic Targets 4 Biological-Templating of a Segregating Binary Alloy for Nanowire-Like Phase-Change Materials and Memory 5 Summary Appendices A1 General Protocols A2 Supplementary Information for Chapter 2 A3 Supplementary Information for Chapter 3 A4 Supplementary Information for Chapter 4 5 Table of Contents 1 INTRODUCTION TO M13 BACTERIOPHAGE, PROTEIN LIBRARIES, & LIGAND SCREENING Overview ............................................................................................................................. 16 M13 Bacteriophage .............................................................................................................. 17 1.2.1 M13 Genome ............................................................................................................................. 18 1.2.2 M13 Capsid Structure ................................................................................................................ 19 1.2.3 M13 Life Cycle with E. coli Host ................................................................................................. 20 Phage Display and Protein Libraries ...................................................................................... 22 1.3.1 Protein Randomization Techniques........................................................................................... 22 1.3.2 Display Technologies ................................................................................................................