Difference between revisions of "Team:Bielefeld-CeBiTec/Results/translational system"

Generating our Toolbox, we used mutated variants of two different aminoacyl tRNA/synthetases (aaRS), tyrosyl-tRNA synthetase and pyrrosylyl-tRNA/synthetase. We proved that the incorporation of non-canonical amino acids (ncaa)through the amber codon is a major metabolic pressure for the cells and therefore imply that the expansion of the genetic code is an efficient approach. In addition to that, we demonstrated that the different aaRS we used in our Toolbox are very different in specificity, and therefore some are not very efficient in incorporating those. This led us to generate our own tyrosyl tRNA‑synthetase library (TyrRS). The TyrRS, based on the wild type Methanococcus jannashii tyrosyl tRNA synthetase , is orthogonal to tRNA/aaRSs from E.coli and suitable to incorporate novel ncAAs. Our library consists of more than 150,000 library plasmids, among them 27,672 different TyrRS variants with variable binding sites. The TyrRS library is the basis for a combination of positive and negative selection cycles, where the different TyrRS variants are selected for specificity incorporating non canonical amino acids. That’s how an optimal adapted TyrRS variant is obtained. We were able to perform both selection cycles, with the Cou- and NPA-amino acid, receiving TyrRS variants being potentially more specific for the incorporation of the certain ncAA