Rewriting the genome of Escherichia coli

Our recently acquired ability to synthesize DNA at large scale is opening the door to writing entire genomes; this constitutes a powerful approach to address fundamental biological questions, and may enable the creation of designer organisms with useful properties. One interesting avenue for investigation is the creation of recoded genomes, where codons are substituted by their synonyms. Compression of synonymous codon boxes may provide blank spaces in the quasi-universal genetic code, and these may be amenable for reassignment into unnatural amino acids, in synergy with parallel efforts to engineer the protein translation machinery. Recoding genomes is subject to both biological and technical challenges. First, synonymous codon choice genome-wide is not trivial, and identifying suitable synonymous replacements is challenging. Second, synthetic DNA pieces need to be assembled into fragments of increasing size, and ultimately implemented inside a target host. Here, recently reported strategies for genome engineering in E. coli (REXER and GENESIS) are extended and used to create a synthetic, recoded E. coli genome. Chapter 2 describes a strategy for assembling large natural genomic DNA pieces into BACs that are substrates for genome replacement. Experiments with these BACs serve to validate and improve REXER and GENESIS, and lay out a strategy for genome replacement. Chapter 3 employs these strategies for the synthesis and assembly of a recoded E. coli genome where all annotated instances of serine codons TCG and TCA, and stop codon TAG, are systematically replaced by their synonyms. The resulting strain, Syn61, provides a unique platform for exploring sense codon reassignment in vivo, and reassignment of the TCG codon to both natural and unnatural amino acids is demonstrated. Finally, Chapter 4 extends the toolkit for genome engineering in E. coli, and provides technologies for splitting the genome into pairs of chromosomes, as well as performing precise inversions and translocations. These technologies are used to precisely combine synthetic sections from distinct strains into a single genome. Together, these technologies may provide a foundation for future genome synthesis endeavours.