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

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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 X possible sequences of an aaRS able to incorporate the new amino acid CBT Asp by modeling.
 
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Revision as of 10:39, 4 October 2017

Toolbox Overview

Analysing

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

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.