Synthesis, Characterisation and Evolution of Escherichia coli with a Compressed Genetic Code

The design and synthesis of entire genomes is a powerful approach that allows the study of biological functions and the creation of new ones: Study-by-synthesis promises a better understanding of the genomic architecture and regulatory mechanisms. Furthermore, synthetic genomes may enable the creation of organisms with new desirable properties. Synthetic genomes with systematic removal of particular codons provide a unique opportunity to investigate rules that govern codon choice and enable the creation of organisms with a compressed genetic code. Such liberated codons can be reassigned to non-canonical amino acids to equip proteins with novel functions. Furthermore, recoded organisms are genetically distinct from all other lifeforms and may therefore prove useful for biocontainment and viral resistance. Chapter I outlines our understanding of the genetic code, intrinsic genomic sequence features such as synonymous codon choice, as well as concepts of genetic code expansion. Furthermore, it elaborates on the design and properties of organisms with synthetic genomes and discusses techniques for large-scale manipulation of genomes. Chapter II describes the total synthesis of Escherichia coli with a recoded genome, in which two sense codons and one stop codon are replaced with synonyms. The chapter details the assembly of BACs with >100 kb of synthetic DNA, the iterative replacement of wild-type genomic sections with a recoded counterpart, the identification and correction of non-tolerated synthetic sequence, and finally the assembly of synthetic genomic regions from partially recoded cells into a single, completely recoded cell named Syn61. Chapter III describes the characterisation of Syn61 and its further development. Multiple rounds of laboratory evolution and deletion of the decoding elements for the removed codons (tRNAs and release factor 1) yielded a cell with a compressed genetic code and improved growth, to advance genetic code expansion. Finally, in Chapter IV the powerful genome engineering method REXER, which enabled synthesis of Syn61, is developed further and a universal platform is established for the integration of any synthetic DNA at a specified locus. Moreover, a method is developed for the combined delivery and integration of synthetic DNA in a simple one-day protocol