Enabling evolution with expanded genetic codes

The natural genetic code is largely shared amongst all terrestrial life, though variations do exist that provide evidence of genetic code evolution. The field of synthetic biology has developed methods to augment and modify the genetic code, by reassigning codons and genetically incorporating noncanonical amino acids (NCAAs). Most commonly, amino-acyl tRNA synthetases and their cognate tRNAs are engineered to recognize (NCAAs), the tRNA charged with an NCAA is then used during translation to decode the amber stop codon. Using this method, over 200 unique NCAAs have been added to the genetic code, allowing for the site-specific incorporation of useful chemistries, including covalent bond formation and fluorescence. NCAAs have been implemented in biological research and protein engineering. The second chapter of this dissertation explores the biophysical parameters that allow efficient crosslinking with a NCAA. We investigated several environmental factors which could impact crosslinking between two interacting proteins. We define a set of parameters that permit efficient crosslinking. Noncanonical amino acids are a potentially powerful tool in understanding genetic code evolution. Evolutionary experiments using NCAAs have already been used to explore proposed theories on codon evolution, including codon capture and ambiguous intermediate theories. Though several studies have explored the implications of NCAAs in evolution, significant obstacles have prevented the long-term evolution of wild-type organisms with expanded genetic codes. In chapter three of this dissertation we demonstrate that a single essential enzyme can be engineered to be structurally dependent on a genetically encoded NCAA. This confers a functional addiction to the NCAA and the addiction can be transferred to new bacterial hosts using a DNA plasmid. Chapter four expands on this idea, attempting to use the chemistries bestowed by the NCAA for active site chemistry. Finally, in chapter five we demonstrate that a protein-conferred cellular NCAA addiction is sufficient for the long-term evolution of bacteria with a recoded amber codon. We investigated the genetic and phenotypic impact of evolution with an expanded genetic code.