Genetic Engineering Toward a 57-Codon Genome The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:37944948 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 © 2016 – Matthieu Landon All rights reserved Dissertation Advisor: George M. Church Matthieu Landon Abstract Scientific progress in fundamental biology has drastically transformed our ability to engineer biological systems, with diverse applications in medicine and industry, from insulin and artemisin production in prokaryotes to gene therapy in eukaryotes. In particular, DNA synthesis technologies and their recent plumetting cost have the potential to transform genome engineering by unlocking unlimited number of edits independent of the parent genome, paving the way for rational genome-wide changes. For example, a powerful strategy for enhancing genomes with functions not commonly found in nature is offered by recoding, the re-purposing of genetic codons. Indeed, since all living organisms share an identical genetic code composed of 64 genetic codons, changing this code allows exploration of new properties such as genetic isolation and expanded protein function. In that context, we set out to explore the feasibility of the construction of an entirely synthetic 57-codon genome in Escherichia coli. Construction of a 57-codon genome is a daunting task, due to the genome scale and to the unprecedented amount of DNA modifications to perform. When I started my PhD, only the UAG stop codon had been successfully replaced genome-wide. To move forward, better knowledge of the consequences of synonymous codon replacements and improved genome design rules were required. Thus, I focused on understanding the consequences of synonymous codon replacements for two sense codons: AGA and AGG. Then, I tackled the validation and troubleshooting of a 57- codon genome in segments, our major effort toward the validation of the entire genome design in E. coli. Last, I developed safe genomically recoded genes to increase containment of recoded organisms, a major concern in the field of synthetic biology. iii Chapter 1 of this dissertation presents an overview of the different current methods for genome engineering as well as their limitations. In particular, it discusses the recent progress in full-genome synthesis, its potential as a radical new way to engineer life and the current limitations to its broad usage. Examples from our recoding efforts are highlighted. Chapter 2 describes our approach to elucidate the rules for genome-wide sense codon replacement on the example of the AGA and AGG sense codons. This chapter is adapted from Napolitano M., Landon M., Gregg C., Lajoie M. et al., Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 113, E5588–97 (2016). Chapter 3 presents our effort to design, synthesize and test a 57-codon genome in E. coli. This chapter shows preliminary evidence that construction of radically recoded genome via synthesis is possible but requires use of a robust pipeline for construction and testing. This chapter is adapted from Ostrov N., Landon M., Guell M., Kuznetsov, G. et al., Design, synthesis, and testing toward a 57-codon genome. Science. 353, 819–822 (2016). Chapter 4 describes a strategy to engineer safe and genetically isolated genes in recoded organisms. This chapter is adapted from a manuscript currently in preparation. However, our efforts to build a 57-codon genome are not yet complete and many more steps have to be accomplished before the entire organism can be constructed. In a final chapter (Chapter 5), I reflect on the next steps toward complete assembly of a 57-codon organism. Appendix A, B and C contain supplemental information (Figures and tables) to Chapter 2, 3 and 4. Appendix D showcases another project that was performed during the course of my PhD. This appendix is adapted from Lukacisin M., Landon M., Jajoo R., (2016). iv Table of contents ABSTRACT .............................................................................................................. III TABLE OF CONTENTS ........................................................................................... V LIST OF FIGURES ................................................................................................. VII LIST OF TABLES ................................................................................................... VII DEDICATION ....................................................................................................... VIII ACKOWLEDGMENTS ............................................................................................ IX CHAPTER 1 LARGE-SCALE DNA SYNTHESIS IN MODIFIED ORGANISMS. .. 1 ABSTRACT .................................................................................................................................... 1 GENOME-EDITING TECHNOLOGIES HAVE TRANSFORMED OUR ABILITY TO MODIFY GENOMES. ....... 2 ALL GENOME-EDITING TECHNOLOGIES HAVE LIMITATIONS. .......................................................... 5 DNA SYNTHESIS OPENS A NEW PARADIGM IN THE FIELD OF GENOME MODIFICATION. .................. 7 WHAT TYPE OF GENOMIC CHANGES ARE THE MOST AMENABLE TO FULL-GENOME SYNTHESIS? ..... 8 WHAT ARE IDEAL CHASSIS FOR DEVELOPING SYNTHETIC ORGANISMS? ....................................... 13 WHAT ARE THE FUTURE BOTTLENECKS OF LARGE-SCALE DNA SYNTHESIS AND ASSEMBLY? ...... 15 CONCLUSION .............................................................................................................................. 19 REFERENCES .............................................................................................................................. 22 CHAPTER 2 EMERGENT RULES FOR CODON CHOICE ELUCIDATED BY EDITING RARE ARGININE CODONS IN ESCHERICHIA COLI ........................ 30 ABSTRACT .................................................................................................................................. 32 MAIN TEXT ................................................................................................................................ 33 MATERIALS AND METHODS ........................................................................................................ 55 SUPPLEMENTAL MATERIAL ........................................................................................................ 61 REFERENCES .............................................................................................................................. 62 CHAPTER 3 DESIGN, SYNTHESIS AND TESTING TOWARD A 57-CODON GENOME ................................................................................................................... 65 ABSTRACT .................................................................................................................................. 66 MAIN TEXT ................................................................................................................................ 67 MATERIALS AND METHODS ........................................................................................................ 78 SUPPLEMENTAL MATERIAL ........................................................................................................ 98 REFERENCES .............................................................................................................................. 99 CHAPTER 4 GENETICALLY ISOLATED GENES FOR WIDE-USE OF NONSTANDARD AMINO ACIDS IN GENOMICALLY RECODED ORGANISMS ................................................................................................................................. 102 ABSTRACT ................................................................................................................................ 103 MAIN TEXT ............................................................................................................................... 104 v FUTURE STEPS ......................................................................................................................... 115 MATERIAL AND METHODS ........................................................................................................ 116 REFERENCES ............................................................................................................................ 123 CHAPTER 5 CONCLUSION AND FUTURE PROJECTS .................................... 125 PLAN FOR COMPLETION OF THE TESTING OF THE 57-CODON GENOME ...................................... 127 GENOME ENGINEERING SOFTWARE IMPROVEMENTS ................................................................ 133 POSSIBLE PLANS FOR HIERARCHICAL ASSEMBLY ....................................................................... 134 FINAL ADJUSTMENTS TO COMPLETE THE STRAIN ...................................................................... 135 GENERAL CONCLUSION ............................................................................................................. 138 REFERENCES ............................................................................................................................ 139 APPENDIX A SUPPLEMENTAL INFORMATION FOR CHAPTER 1 .............. 140 SUPPLEMENTARY FIGURES ......................................................................................................