Our aim was to provide an innovative toolbox to incorporate different noncanonical amino acids (ncAAs) during translation to expand the possibilities in protein design. We choose seven different ncAAs in our five tools and demonstrate them all in interesting applications. These ncAAs can be used for a lot of different approaches in basic research, medicine and manufacturing and with our submitted parts, every iGEM team can incorporate these seven ncAAs into their target proteins. A short description of every tool is listed below.
Analysing
The structural analysis of a protein can be used to study distances between noncanonical amino acids (ncAAs) with Foerster Resonance Energy Transfer (FRET). This provides measuring distances between specific incorporated amino acids in the target protein. To incorporate the ncAAs we provide three different tRNA/aminoacyl-synthetases which incorporate in respose to the amber or the less used leucine codon. To demonstrate this tool we also developed a prion detection assay.
Photoswichting
We designed the concept of a light influenced lycopene production as basis for the establishment of a photo switchable enzyme activity. We introduced an amber-codon in the gene for crtI in the lycopene pathway and thus stopped the lycopene production. We then showed that the lycopene production can be partially retained, by cotransforming with an aaRS and cultivation without the matching ncAA.
Labeling
The labeling of a protein in vivo is a useful tool that allows the investigation of a protein in its native environment. As a label for our target protein we use the fluorescent amino acid L-(7 hydroxycoumarin 4 yl) ethylglycine (CouAA) that is incorporated by an orthogonal t-RNA/aminoacyl synthethase pair (tRNA/aaRS) at a defined position. We provide this tRNA/aaRS to the iGEM community and demonstrated this tool with the colocalization of the artificial cell compartment carboxysome and the ribulose 1,5-bisphosphate carboxylase oxygenase.
Photolysis
We designed an aminoacyl-tRNA-syntethase (aaRS) for the incorporation of 2-nitrophenylalanine (2-NPA) based on an experiment from Liu et al. (2009) and cloned it into both the pSB1C3 to send it to the head quarter and in pSB3K5 for further characterization. We also designed a fusion protein of GFP and streptavidin with a linker containing an amber codon and showed that only the GFP unit is expressed under normal conditions.The whole fusion protein is produced if cotransformed with the 2-NPA-RS.
Fusing
We explored the possibilities of a new method for highly specific protein terminus independent fusing based on the binding of 1,2‑aminothiols and the cyano group of cyanobenzothiazole (CBT). Therefore, we designed and synthetized a whole new amino acid by coupling the amino group of 6 amino-2-cyanobenzothiazole and the carboxyl group of the side chain of aspartic acid. The result was Nγ 2 cyanobenzothiazol 6 yl L asparagine (CBT-Asp). As counterpart, we used Nε-L-cysteinyl-L-lysine (CL), which contains the 1,2-aminothiol group. This way, we created a pair of non-canonical amino acids which residues can bind covalently to each other under physiological conditions. We cloned the aaRS for CL in pSB1C3 and predicted 12 possible sequences of an aaRS able to incorporate the new amino acid CBT Asp by modeling.
