| dc.description.abstract | The genetic code normally uses the canonical twenty amino acids in order to construct proteins and facilitate life. The process of translation involves an RNA template and codons that will be read and matched to corresponding tRNA molecules carrying charged amino acids. An aminoacyl tRNA synthetase (aaRS) specific to each amino acid is responsible for loading and charging the correct amino acid to the tRNA. In recent years, a few orthogonal pairs of the tRNA and aaRS have been utilized to expand the genetic code past the natural 20 amino acids. Expanding the genetic code can provide new insight into protein function, structure, and interactions within the cell. The introduction of new amino acids could lead to proteins with new chemical or biological activity and even advantageously alter function leading to evolutionary events. In our research, we attempt to incorporate unnatural amino acids using an orthogonal pair of Methanobacterium thermoautotrophicum leucyl-tRNA synthetase (MLRS) and Halobacterium sp. Leucyl tRNA. A mutant MLRS lacking an editing domain (MLRS DCP1) was generated. The best variant was isolated and sequenced. The leucine binding site, determined from structural homology, was randomized at five positions to create a library of mutants. In the positive selection, only the cells containing the MLRS DCP1 variants that add an amino acid to the tRNA will survive in the presence of chloramphenicol. In the negative selection, the cells containing the variants that add natural amino acids to the tRNA will die in the presence of 5-fluorouracil. The library can then be used for further experiments to determine how effectively unnatural amino acids are incorporated. | |
