Team:Bielefeld-CeBiTec/Project/translational system

Translational System Overview

The whole expression of proteins is based on the DNA coded in the genetic triplet code. Therefore, this code is essential for all living organisms. The expansion of the genetic code, by adding new bases to the DNA, offers multiple possibilities, like the in vivo integration of ncAA in proteins in vivo. At the same time, new challenges arise constantly. Besides ensuring correct replication and preservation of the unnatural-base pairs (UBP) , the whole transcriptional and translational system has to be adapted if a non-canonical amino acid (ncAA) is coded by UBPs. The incorporation of ncAA generates many new properties of proteins and also a wide specter of applications. It can be achieved either by the use of UBPs or coded by an amber-stop codon.
The incorporation of a ncAA by an amber-stop codon requires the recognition of the codon and a tRNA/aminoacyl-synthetase (tRNA/aaRS) pair which is able to accept and bind the ncAA (to charge the tRNA with the ncAA). In addition, the tRNA/aaRS pair should be very specific in the incorporation of the ncAA. Therefore a library of the mutated orthogonal tRNA/aaRS is generated and undergoes numerous rounds of positive and negative selection cycles for the adaption of the codon recognition and the amino acid binding. The selection results in a synthetase which can be expressed efficiently in E. coli and is able to reliable incorporate an ncAA