Steven Stanley, Brigham Young University
The genetic code has long been restricted to a set of 20 fundamental building blocks called amino acids. Recent research has expanded the genetic code through unnatural amino acids (uAA), thus adding enormous possibilities to the potential chemistries of proteins. Because nature is highly selective in the protein translation process, it has proven extremely difficult to successfully insert multiple uAAs simultaneously. The incorporation of an uAA with in vitro methods typically relies on the use of the amber stop codon as a mutated insertion site. A stop codon placed at the middle of a gene can code for either the uAA or termination, thus, protein synthesis may often terminate prematurely instead of inserting the desired uAA. This inefficiency inhibits the possibility of inserting multiple uAAs simultaneously. We propose a novel method that will allow for multiple uAAs to be inserted simultaneously. Our method involves isolating a minimal set of tRNA for in vitro protein synthesis, allowing for uAA insertion to occur at codons other than the amber stop codon. My work has focused on the production of 4 versions of uAA-tRNA synthetase, a protein that charges tRNA with the uAA. We are currently growing these 4 different proteins in bulk and testing their activity. We will test them for compatibility, confirming that they do not interfere with one another and other synthetases native to our in vitro protein synthesis system. These uAA-tRNA synthetases, in conjunction with specialized tRNA, will provide the basis to efficiently incorporate multiple uAA simultaneously. The success of this project will have many practical applications ranging from new therapeutics to new methods of medical diagnosis.