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Generalization of Genetic Code Expansion Generalization of Genetic Code Expansion The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Stork, Devon. 2020. Generalization of Genetic Code Expansion. Doctoral dissertation, Harvard University Graduate School of Arts and Sciences. Citable link https://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37368951 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA HARVARD UNIVERSITY Graduate School of Arts and Sciences DISSERTATION ACCEPTANCE CERTIFICATE The undersigned, appointed by the Department of Molecular and Cellular Biology have examined a dissertation entitled Generalization of Genetic Code Expansion presented by Devon Stork candidate for the degree of Doctor of Philosophy and hereby certify that it is worthy of acceptance. Signature Richard Losick (Sep 15, 2020 15:40 EDT) Typed name: Prof. Richard Losick Vlad Denic Signatur Vlad Denic (Sep 17, 2020 14:52 EDT) Typed name: Prof. Vladimir Denic Signature Abhishek Chatterjee (Sep 23, 2020 13:28 EDT) Typed name: Prof. Abhishek Chatterjee Signature Typed name: Prof. Signature Typed name: Prof. Date: September 15, 2020 Generalization of Genetic Code Expansion A dissertation presented by Devon Stork to The Department of Molecular and Cellular Biology In partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Biochemistry Harvard University Cambridge, Massachusetts September 2020 © 2020 Devon Stork All rights reserved Dissertation Advisors: Dr. Ethan Garner and Dr. George Church Devon Stork Generalization of Genetic Code Expansion ABSTRACT The standard genetic code directs the assembly of the 20 standard amino acids into proteins and defines function in biology. Through the central dogma, DNA is transcribed into RNA which is translated by the well-understood machinery of the ribosome and accompanying tRNA, using the genetic code to create the proteins that accomplish most tasks in life. The field of genetic code expansion has focused on incorporating synthetic ‘non-standard amino acids’ (nsAAs) with novel chemical structures into the genetic code. This is done by engineering an aminoacyl-tRNA synthetase to conjugate an externally provided nsAA onto an engineered tRNA in vivo such that it will proceed to the ribosome for standard translation, being incorporated into a growing polypeptide chain. Once incorporation has been achieved, nsAAs allow for site- specific encoding of a defined chemical function, without the limitations of the standard genetic code or the requirement of complex protein engineering. With over 150 nsAAs demonstrated in the literature, a broad array of functions are available for experiment and application. However, the contexts in which they can be used are limited. In this thesis, I investigate ways to broaden the applications of existing genetic code expansion tools. I begin with a description of a post-translational proofreading tool capable of distinguishing between proteins successfully charged with a ‘correct’ nsAA and proteins with an ‘incorrect’ nsAA or standard amino acid. We repurposed a natural protein degradation iii pathway, the N-end rule, to degrade proteins that were not properly charged with the target nsAA. This system could be tuned by engineering an adaptor protein to change the desired nsAA profile, allowing different versions of post-translational proofreading to check for distinct nsAAs. Finally, we demonstrated that this tool improved the purity of desired product for promiscuous genetic code expansion systems and facilitated the directed evolution of more specific genetic code expansion systems. Next, I explore genetic code expansion beyond the optimal conditions of strains specifically engineered to enhance nsAA incorporation. My coauthors and I investigate the use of peptides derived from honeybee antimicrobial molecules which could transiently inhibit competition with genetic code expansion. These peptides allow improved nsAA incorporation into various biotechnologically relevant E. coli strains as well as facilitate the expansion of the Agrobacterium tumefaciens genetic code for the first time. Finally, I apply the tools of genetic code expansion to the bacteria Bacillus subtilis and demonstrate that nearly any nsAA used in E. coli can be applied in B. subtilis using identical synthetases. I explain that nsAA incorporation into native stop codons is much more common than in E. coli, suggesting differences in translational termination between the two organisms. I also utilize nsAAs for translational titration and photocrosslinking in B. subtilis, showing that these tools can be easily utilized for novel kinds of experiments. Together, these tools will help expand the scope of genetic code expansion beyond specifically engineered strains and nsAAs. iv Acknowledgements This thesis and all the work behind it would not have been possible without a huge amount of effort on the part of others in training and supporting me. As a graduate student, I’ve received quality mentoring from many people, professor, postdoc and graduate student. My advisors Ethan Garner and George Church each supported and mentored me in their own way, and I am thankful for the hands-off but responsive advising style. I was allowed to wander until lost and then helped to find my place again. They also encouraged to follow my interests wherever they lead me, even if that was outside of academia. I also thank my committee, Rich Losick, Abhishek Chatterjee and Vlad Denic for their helpful advice and support. I owe much to the various postdocs I’ve interacted with over the years. Most notably Aditya Kunjapur, who took me under his wing when my project was going nowhere and has had so much to teach me about genetic code expansion, professionalism, and the true scientific process. We’ve continued collaborating as he became a professor and I wait with bated breath to see what his lab will accomplish with these technologies. I’ve also worked closely with Erkin Kuru, an irrepressible spirit of unrivalled creativity and brilliance. I believe in his projects even when they don’t work the first time. Many other postdocs have given me invaluable advice and feedback, including Alex Bisson, Cory Smith, Jorge Marchand, and Kamesh Narasimhan. My fellow students have provided an important feeling of camaraderie. My MCO class doing homework together was vital for the first years of graduate school, and staying in touch through D&D has been an important place to socialize. I still owe Mary Morrison, Korleki Akiti and Andrew Kane the finale to the story of the Dragonriders. The fellow students in the Garner v and Church labs have been great people to complain about science to and get advice on tough experiments. Georgia Squyres has been a role model of mine for her incredible way of analyzing the literature and planning good experiments since my second year. Max Shubert, Gabe Filsinger, George Chao and Sean Wilson have been important sounding boards and sources of support for failed experiments. Outside of the lab, I’ve had a great deal of support from friends, family and previous educators. My parents and especially my father, Christof Stork, encouraged my curiosity and interest in science from a young age, while my mother Terri Olson kept it tempered by practicality. I’ve had such a wonderful series of amazing teachers at every level of education that I really do wonder if I can take any credit for what I’ve learned. My long-term friends Andrew Gibiansky and Conrad de Kerkhove have shaped my views of the world, even if we only spend time together intermittently. Sarah Scheffler has been an incredible best friend and I’ve looked forward to every time we hang out. Finally, my partner Amanda Lemire has been my daily source of support, conversation, and fortitude, and I cannot thank her enough for helping me find my center even on bad days. vi Table of contents ABSTRACT ............................................................................................................................ iii Acknowledgements ............................................................................................................... v Table of contents ................................................................................................................. vii List of Figures ....................................................................................................................... ix List of Tables ......................................................................................................................... x Abbreviations ....................................................................................................................... xi Scientific contributions to this Thesis:....................................................................................xii Chapter 1: Introduction.......................................................................................................... 1 1.1 Genetic Code Expansion ...........................................................................................................1 1.2 Engineered tRNA & tRNA-synthetase diversity ..........................................................................4 1.3 Orthogonality and specificity of genetic code expansion ............................................................6
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