Global Incorporation of Unnatural Amino Acids in <i>Escherichia coli</i>

The incorporation of amino acid analogs is becoming increasingly useful. Site-specific incorporation of unnatural amino acids allows the application of chemical biology to protein-specific investigations and applications. However, the global incorporation of unnatural amino acids allows for tests of proteomic and genetic code hypotheses. For example, the adaptation of organisms to unnatural amino acids may lead to new genetic codes. To understand and quantify changes from such perturbations, an understanding is required of the microbiological and proteomic responses to the incorporation of unnatural amino acids. Here we describe protocols to characterize the effects of such proteome-wide perturbations.