- Analyzing (BBa_K2201201, BBa_K2201202): Two different ncAAs are incorporated which are labeled with fluorophores in a chemically reaction. With the help of Foerster Resonance Energy Transfer (FRET), the distance between the amino acids could be measured, to get information about the conformation.
- Photoswitching (BBa_K2201207): A photoisomerisable amino acid could be incorporated which changes its conformation when it is irradiated with light of different wavelengths. This conformation change can be used to inhibit the functionality of the target protein, so that reactions can be switched on and off on the protein level using only light exposure.
- Labeling (BBa_K2201204): A fluorescent amino acid could be used to label the target protein in vivo. The advantages compared to fluorescent proteins lay in the smaller size of the fluorescent amino acid.
- Photolysis (BBa_K2201200): Photolysis amino acids break the peptide backbone at the position they were incorporated. This cleaving could be used to activate or deactivate proteins.
- Fusing (BBa_K2201208, BBa_K2201302): Two ncAAs which form a specific covalent bond between each other can be used to fuse proteins together or immobilize the target protein, independent of the C- or N-termini.
We hope that our toolbox will be used frequently by future iGEM-teams and help them at their projects. We also wish that they will add new synthetases for other ncAAs and thus expand the possibilities of further applications in protein design.
Two Different Ways on how to Incorporate ncAAs
Figure 2: Expanded code sun. Representation of the code sun after the incorporation of one new unnatural base to expand the genetic code and create new blank codons (purple) that can be used to evolve aaRS to enable ncAAs for advanced protein design.