Whole genome sequencing constitutes one of the hallmark advances of biological sciences over the last decade. Genome scale engineering -- the capacity to engineer hundreds or thousands of changes into a genome -- will constitute a critical technical advance over the coming ten years. This project seeks to develop a set of tools and technologies to re-engineer the bacterial genome of Escherichia coli. In the first phase the project seeks to recode all instances of the rarest stop codons (amber, UAG) to the most common stop codon (UAA), creating a free completely unused codon which can be repurposed for a number of applications, including the introduction of non-natural amino acids and for applications in protein based therapeutics. This goal can be accomplished by introducing approximately 300 specific point mutants at sites widely distributed throughout the genome. Later phases will pursue more extensive codon reassignments, constituting thousands of changes to the genome. These types of large scale, broadly distributed modifications to an organism have not been realized previously and represent a fundamental re-structuring of the genetic code. A central goal of such genetic code engineering is to advance the understanding of the structure of biology's operating system, to gain new insights into existing organisms as well as into how to engineer new biological systems. It is anticipated that the new strains generated in this work will be made available to the broader biomedical and biotechnology communities. These will be especially useful for incorporating non-natural amino acids into proteins, facilitating the manufacture of enzymes and other new biologically based materials. Furthermore, the technologies developed to achieve these objectives should comprise a set of tools to allow researchers to implement new genome-scale engineering projects that were not possible before.
