Difference between revisions of "Team:Bielefeld-CeBiTec/Demonstrate"

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<b>Writing a <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/HP/Silver">biosafety report</a> titled “Auxotrophy to Xeno-DNA: A Comprehensive Exploration of Combinatorial Mechanisms for a High-Fidelity Biosafety System” </b>
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<b>Writing a <a target="_blank" href="https://static.igem.org/mediawiki/2017/1/1e/T--Bielefeld-CeBiTec--DKE_Biosafety_Report.pdf">biosafety report</a> titled “Auxotrophy to Xeno-DNA: A Comprehensive Exploration of Combinatorial Mechanisms for a High-Fidelity Biosafety System” </b>
 
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Revision as of 21:59, 1 November 2017

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 expand the possibilities in protein design. We incorporated a trinucleotide transporter to enable E. coli to take up the unnatural bases iso-dGTP and iso-CmTP, thus these could be incorporated. Furthermore we identified an enzyme from the native iso-dGTP producent Croton tiglium for the bio-synthesis of this unnatural base in E. coli. Further we showed that certain Taq polymerases can incorporate these bases in vitro. To sequence the unnatural base pairs, we also developed two orthogonal methods to detect the unnatural bases in our target sequence and programmed a software for both. First a nanopore sequencing method and a restriction experiment.

To use these new codons, we developed a library of over eight-thousand synthetase sequences and a positive/negative selection system to obtain new aminoacyl-tRNA-synthetases. These can be applied to charge non-canonical amino acids to the tRNA against the novel codon 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. We also improved a test system and defined a ranking system for aminoacyl-tRNA synthetases

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!

Achievements


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



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


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



Modeling more than ten new aaRS sequences



Library development with several hundred 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-well microtiter plates, which can be used to manipulate non-canonical amino acids and much more



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


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



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