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

Revision as of 20:22, 31 October 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

While we were not able to incorporate non-canonical amino acids through semi-synthetic codons, 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

Proof that certain Taq-polymerases can efficiently incorporate unnatural nucleotides

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

Development of a photoswitchable lycopene pathway

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”

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 Our goal was to introduce <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/unnatural_base_pair">unnatural bases</a></b> into the genetic code of <i>E.coli</i> to create new blank codons for the translational incorporation of <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/toolbox">non-canonical amino acids</a></b> to expand the possibilities in protein design.
-We incorporated a <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/unnatural_base_pair/uptake_and_biosynthesis">trinucleotide transporter</a></b> to enable <i>E. coli </i> 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 <i>Croton tiglium</i> for the <b><a target="_blanl" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/unnatural_base_pair/biosynthesis">trinucleotide transporter</a></b>.">bio-synthesis of this unnatural base</a></b> in <i>E. coli</i>. Further we showed that certain Taq polymerases can incorporate these bases <i>in vitro</i>. 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 <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Software">nanopore sequencing method</a></b> and a <b>restriction experiment</b>. <br><br>
+We incorporated a <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/unnatural_base_pair/uptake_and_biosynthesis">trinucleotide transporter</a></b> to enable <i>E. coli </i> 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 <i>Croton tiglium</i> for the <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/unnatural_base_pair/biosynthesis">bio-synthesis of this unnatural base</a></b> in <i>E. coli</i>. Further we showed that certain Taq polymerases can incorporate these bases <i>in vitro</i>. 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 <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Software">nanopore sequencing method</a></b> and a <b>restriction experiment</b>. <br><br>
 To use these new codons, we developed a <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/translational_system/library_and_selection">library</a></b> of over eight-thousand synthetase sequences and a positive/negative <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/translational_system/library_and_selection">selection system</a></b> to obtain new <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox">aminoacyl-tRNA-synthetases</a></b>. These can be applied to charge non-canonical amino acids to the tRNA against the novel codon and to turn <b>semi-synthetic codons</b> functional. <br><br>
 To demonstrate the benefits of non-canonical amino acids to the synthetic biology community, we worked on five <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/toolbox">applications</a></b> utilizing non-canonical amino acids. Furthermore, we designed and synthetized our own fully <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/toolbox/fusing">synthetic non-canonical amino acid</a></b> and <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Model">modeled</a></b> possible synthetase-sequences for its incorporation. We also improved a <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Improve">test system</a></b> and defined a ranking system for aminoacyl-tRNA synthetases<br><br>