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Revision as of 08:05, 27 October 2017

iGEM Bielefeld-CeBiTec 2017

Expanding the Genetic Code

We are exploring the application of unnatural base pairs as an expansion of the genetic code. To prevent loss of unnatural base pairs during replication, we will utilize several systems including CRISPR/Cas9. The expanded genetic code allows for the ribosomal incorporation of multiple non-canonical amino acids (ncAAs) into peptides. With their broad chemical and functional diversity, ncAAs provide a variety of promising applications including protein labeling, photocaging, structure analysis, and specific protein interactions. Therefore, our innovative toolkit for the translational incorporation of ncAAs in E. coli is a valuable contribution to iGEM. Directed evolution of tRNA/aminoacyl-tRNA synthetase pairs enables the site-specific incorporation of ncAAs into peptides. This approach results in an optimal orthogonality to the autologous translation apparatus and a high flexibility concerning the incorporation of multiple ncAAs. As proof of concept, we are developing a rapid test for prions and a new chromatography method for mild protein elution.
For more information visit our project poster on reasearch gate

Establishment of two orthogonal methods for the detection of unnatural base pairs in a target sequence via Oxford Nanopore sequencing and an enzyme based detection method

Development of a software suite for these orthogonal methods

Integration and characterization of the nucleotide transporter PtNTT2 from P.tricornutum in E.coli for the uptake of unnatural nucleoside triphosphates

Proof that certain Taq-polymerases can efficiently incorporate unnatural nucleotides

Construction of a toolkit consisting of five aminoacyl-tRNA synthetases for incorporation of non-canonical amino acids

Development of a photoswitchable lycopene pathway

Design and chemical synthesis of a novel, fully synthetic amino acid based on cyanonitrobenzothiazol and asparagine and proof of its functionality

Modeling more than ten new aaRS sequences

Library development with several thousand sequences for selecting aminoacyl-tRNA synthetases

Construction of positive and negative selection plasmids for the evolution of new synthetases for non-canonical amino acids

Improvement of an aminoacyl-tRNA synthetase test-system by introducing a FRET-system and development of a ranking system

Construction of an LED panel for irradiating 96-microwell plates, which can be used to manipulate non-canonical amino acids and much more

Development of an Androuid App to control the LED panel with your smartphone via Bluetooth

Writing of a biosafety report titled “Auxotrophy to Xeno-DNA: A Comprehensive Exploration of Combinatorial Mechanisms for a High-Fidelity Biosafety System”

Writing of the ChImp Report on the “Chances and Implications of an Expanded Genetic Code”