Team:Bielefeld-CeBiTec/Demonstrate

Demonstrate Your Work
Our goal was to introduce unnatural bases into the genetic code of E.coli to create new blank codons for the translational incorporation of non-canonical amino acids. To demonstrate that the incorporation of unnatural base pairs is possible, we demonstrated that E. coli can take up iso-dGTP and iso-CmTP from the media through a nucleotide transporter. Further we showed that certain Taq polymerases can incorporate these bases in vitro. To sequence the unnatural base pairs, we also developed a nanopore software modification and were able to proof the presence of unnatural base pairs using our modified software and by a simple restriction experiment developed by us.

To use these new blank codons, we developed a library several thousand synthetase sequences and a positive/negative selection system to obtain new aminoacyl-tRNA-synthetases. These can be applied to couple non-canonical amino acids to the tRNA and to turn semi-synthetic codons functional.

To demonstrate the benefits of non-canonical amino acids to the synthetic biology community, we worked on five applications utilizing non-canonical amino acids. Furthermore, we designed and synthetized our own fully synthetic non-canonical amino acid and modeled possible synthetase-sequences for its incorporation.

While we were not able to incorporate non-canonical amino acids through semi-synthetic codons, we are convinced that we have laid the foundations for a whole new field of synthetic biology for the iGEM community. We would be very honored if future teams would build on our project to further develop this approach and to develop new and exciting applications! Expand!