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

 
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Demonstrate Your Work
 
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<h2>Demonstrate your Work</h2>
 
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Our goal was to introduce <b>unnatural bases</b> into the genetic code of <i>E.coli</i> to create new blank codons for the translational incorporation of <b>non-canonical amino acids</b>. To demonstrate that the incorporation of unnatural base pairs is possible and that <i>E. coli</i> can take up iso-dGTP and iso-CmTP from the media through a <b>nucleotide transporter</b>. Further we showed that certain Taq polymerases can incorporate these bases <i>in vitro</i>. To sequence the unnatural base pairs, we also developed a <b>nanopore software</b> modification and were able to proof the presence of unnatural base pairs using our modified software and by a simple <b>restriction experiment</b> developed by us. <br><br>
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We worked in many different scientific fields to find suitable ways for the translational incorporate of non-canonical amino acids into proteins. Repurposing existing codons or incorporating new bases are two possible ways. We realized both ways to expand the genetic code of <i>Escherichia coli.</i> <br><br>
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To use these new blank codons, we developed a <b>library</b> several thousand synthetase sequences and a positive/negative <b>selection system</b> to obtain new <b>aminoacyl-tRNA-synthetases</b>. These can be applied to couple non-canonical amino acids to the tRNA and to turn <b>semi-synthetic codons</b> functional. <br><br>
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The repurposing of a codon for the incorporation of a non-canonical amino acid (ncAA) is possible using the rarely used amber stop codon UAG or other rarely used codons like the leucine codon CUA. To incorporate a non-canonical amino acid using these codons, an orthogonal tRNA/aminoacyl-tRNA synthetase (<b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/translational_system">tRNA/aaRS</a></b>) pair is necessary, which can charge the ncAA to the tRNA. We designed and synthetized the novel ncAA N<sup>γ</sup>&#x2011;2&#x2011;cyanobenzothiazol&#x2011;6&#x2011;yl&#x2011;L&#x2011;asparagine (<b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Applied_Design">CBT-asparagine</a></b>). This ncAA has the chemical ability of perform a highly specific covalent binding reaction, which we wanted to incorporate into our target protein. Therefore, we created a <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/translational_system/library_and_selection">library</a></b> of aaRS with random mutagenized amino acid binding sites and a <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/translational_system/library_and_selection">selection system</a></b> to select for the aaRS that specifically incorporates the ncAA. In parallel to the libary and selection based approach, we <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Model">modeled</a></b> the aaRS which could incorporate our new amino acid CBT-asparagine. We demonstrated that both ways are suitable for the evolution of aaRS. <br><br>
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To demonstrate the benefits of non-canonical amino acids to the synthetic biology community, we worked on five <b>applications</b> utilizing non-canonical amino acids. Furthermore, we designed and synthetized our own fully <b>synthetic non-canonical amino acid</b> and <b>modeled</b> possible synthetase-sequences for its incorporation. We also improved a <b>test system</b> and defined a ranking system for aminoacyl-tRNA synthetases<br><br>
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Although incorporation of ncAAs through the amber codon works, there are challenges associated with this approach. The repurposing of codons leads to the decrease of the growth rate of E. coli and it is only feasible to incorporate up to two different ncAAs. Therefore, we took a new way to incorporate ncAAs. The incorporation of an <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/unnatural_base_pair">unnatural base pair</a></b> into the DNA generates 64 new codons. Our first challenge was the uptake of the unnatural base from the media, because <i>E.coli</i> has no nucleoside triphosphate transporter and is not able to synthetize the bases itself. We cloned a nucleoside <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/unnatural_base_pair/uptake">triphosphate transporter</a></b> that enables the uptake of both bases from the media. Furthermore, we analyzed the transcriptome  of the plant <i>Croton tiglium</i>, which produces the unnatural base isoG. The transcriptome revealed an enzyme for the biosynthesis, which was  cloned and characterized for the <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/unnatural_base_pair/biosynthesis">biosynthesis</a></b> of isoG in <i>E. coli</i>. To <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/unnatural_base_pair/development_of_new_methods">detect</a></b> the unnatural base we developed two orthogonal systems. A restriction experiment based on the <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/unnatural_base_pair/development_of_new_methods">software tool M.A.X</a></b>. and an adaption of the <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/unnatural_base_pair/development_of_new_methods">Oxford Nanopore sequencing</a></b>, which were combined into one <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Software">software suite</a></b>. <br><br>
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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! <b>Expand</b>!
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To demonstrate the possibilities offered by the incorporation of ncAAs, we developed a <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/toolbox">toolbox</a></b> containing five different tools. We chose seven different ncAAs for these five tools and demonstrated interesting applications for them. These ncAAs can be used for various approaches in basic research, medicine and manufacturing. Furthermore, with our submitted <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Parts">parts</a></b>, every iGEM team can incorporate these ncAAs into their target proteins.<br><br>
−
 
+
Regarding our project, two of the ncAAs that are part of our toolbox perform an autocatalytic reaction upon irradiation with ultraviolet light. Therefore, we decided to build our own <b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Hardware">LED panel</a></b> that allows us to perform experiments with these non‑canonical amino acids under reproducible irradiation conditions.
 
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<h2>Achievements</h2>
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<b>We established two orthogonal methods for the detection of unnatural base pairs in a target sequence: an <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Software">Oxford Nanopore sequencing</a> application and an enzyme based detection method</b>
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<article><br><br>
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<b>Development of a <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Software">software</a> suite for these orthogonal methods </b>
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</article>
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<p class="figure subtitle"></p>
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<article><br>
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<b>Integration and characterization of  the <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/unnatural_base_pair/uptake_and_biosynthesis">nucleotide transporter PtNTT2</a> from <i>P.tricornutum</i> in <i>E.coli</i> for the uptake of  unnatural nucleoside triphosphates</b>
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/9/94/T--Bielefeld-CeBiTec--YKE_Bingo.png">
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<p class="figure subtitle"></p>
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</div>
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<div class="third double">
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<article><br><br>
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<b>Confirmation that certain Taq DNA polymerases can efficiently <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/unnatural_base_pair/preservation_system">incorporate unnatural nucleotides</a> </b>
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/9/94/T--Bielefeld-CeBiTec--YKE_Bingo.png">
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<p class="figure subtitle"></p>
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</div>
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</div>
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<div class="third double">
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<article><br><br>
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<b>Construction of a <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/toolbox">toolbox</a> consisting of five aminoacyl-tRNA synthetases for incorporation of non-canonical amino acids</b>
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/9/94/T--Bielefeld-CeBiTec--YKE_Bingo.png">
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<p class="figure subtitle"></p>
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</div>
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<div class="third double">
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<article><br><br>
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<b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/toolbox/labeling">Colocalization</a> of the RuBisCo and and subcellular compartment (carboxysome) using a fluorescent amino acid</b>
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<p class="figure subtitle"></p>
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<div class="third double">
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<article><br><br>
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<b>Development of a <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/toolbox/photoswitching">photoswitchable lycopene pathway</a></b>
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<p class="figure subtitle"></p>
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<div class="third double">
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<article><br>
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<b>Design, <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/toolbox/fusing">chemical synthesis</a> and proof of functionality of a novel, fully synthetic amino acid based on cyanonitrobenzothiazol and asparagine</b>
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</article>
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<div class="contentline">
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<div class="figure small">
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/9/94/T--Bielefeld-CeBiTec--YKE_Bingo.png">
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<p class="figure subtitle"></p>
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</div>
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</div>
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<div class="third double">
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<article><br><br>
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<b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Model">Modeling</a> more than ten new aaRS sequences</b>
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</article>
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</div>
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<div class="contentline">
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/9/94/T--Bielefeld-CeBiTec--YKE_Bingo.png">
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<p class="figure subtitle"></p>
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<div class="third double">
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<article><br><br>
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<b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/translational_system/library_and_selection">Library development</a> with several hundred thousand sequences for selecting aminoacyl-tRNA synthetases</b>
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<div class="contentline">
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/9/94/T--Bielefeld-CeBiTec--YKE_Bingo.png">
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<p class="figure subtitle"></p>
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</div>
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</div>
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<div class="third double">
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<article><br>
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<b>Construction of positive and negative <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/translational_system/library_and_selection">selection plasmids</a> for the evolution of new synthetases for non-canonical amino acids</b>
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/9/94/T--Bielefeld-CeBiTec--YKE_Bingo.png">
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<p class="figure subtitle"></p>
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<div class="third double">
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<article><br><br>
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<b><a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Improve">Improvement</a> of an aminoacyl-tRNA synthetase test-system by introducing a FRET-system and development of a ranking system</b>
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/9/94/T--Bielefeld-CeBiTec--YKE_Bingo.png">
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<p class="figure subtitle"></p>
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</div>
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</div>
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<div class="third double">
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<article><br><br>
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<b>Construction of an <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Hardware">LED panel</a> for irradiating 96-well microtiter plates, which can be used to manipulate non-canonical amino acids and for other applications  </b>
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/9/94/T--Bielefeld-CeBiTec--YKE_Bingo.png">
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<p class="figure subtitle"></p>
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<article><br><br>
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<b>Development of an <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Software">Android</a> App to control the LED panel with your smartphone via Bluetooth</b>
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<p class="figure subtitle"></p>
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<article><br>
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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> entitled “Auxotrophy to Xeno-DNA: A Comprehensive Exploration of Combinatorial Mechanisms for a High-Fidelity Biosafety System” </b>
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<div class="third">
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<div class="figure small">
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/9/94/T--Bielefeld-CeBiTec--YKE_Bingo.png">
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<p class="figure subtitle"></p>
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</div>
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</div>
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<div class="third double">
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<article><br><br>
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<b>Writing the <a target="_blank" href="https://static.igem.org/mediawiki/2017/1/18/T--Bielefeld-CeBiTec--CMZ-ChImp.pdf">ChImp Report</a> on “Chances and Implications of an Expanded Genetic Code”</b>
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Latest revision as of 03:44, 2 November 2017

Demonstrate your Work

We worked in many different scientific fields to find suitable ways for the translational incorporate of non-canonical amino acids into proteins. Repurposing existing codons or incorporating new bases are two possible ways. We realized both ways to expand the genetic code of Escherichia coli.

The repurposing of a codon for the incorporation of a non-canonical amino acid (ncAA) is possible using the rarely used amber stop codon UAG or other rarely used codons like the leucine codon CUA. To incorporate a non-canonical amino acid using these codons, an orthogonal tRNA/aminoacyl-tRNA synthetase (tRNA/aaRS) pair is necessary, which can charge the ncAA to the tRNA. We designed and synthetized the novel ncAA Nγ‑2‑cyanobenzothiazol‑6‑yl‑L‑asparagine (CBT-asparagine). This ncAA has the chemical ability of perform a highly specific covalent binding reaction, which we wanted to incorporate into our target protein. Therefore, we created a library of aaRS with random mutagenized amino acid binding sites and a selection system to select for the aaRS that specifically incorporates the ncAA. In parallel to the libary and selection based approach, we modeled the aaRS which could incorporate our new amino acid CBT-asparagine. We demonstrated that both ways are suitable for the evolution of aaRS.

Although incorporation of ncAAs through the amber codon works, there are challenges associated with this approach. The repurposing of codons leads to the decrease of the growth rate of E. coli and it is only feasible to incorporate up to two different ncAAs. Therefore, we took a new way to incorporate ncAAs. The incorporation of an unnatural base pair into the DNA generates 64 new codons. Our first challenge was the uptake of the unnatural base from the media, because E.coli has no nucleoside triphosphate transporter and is not able to synthetize the bases itself. We cloned a nucleoside triphosphate transporter that enables the uptake of both bases from the media. Furthermore, we analyzed the transcriptome of the plant Croton tiglium, which produces the unnatural base isoG. The transcriptome revealed an enzyme for the biosynthesis, which was cloned and characterized for the biosynthesis of isoG in E. coli. To detect the unnatural base we developed two orthogonal systems. A restriction experiment based on the software tool M.A.X. and an adaption of the Oxford Nanopore sequencing, which were combined into one software suite.

To demonstrate the possibilities offered by the incorporation of ncAAs, we developed a toolbox containing five different tools. We chose seven different ncAAs for these five tools and demonstrated interesting applications for them. These ncAAs can be used for various approaches in basic research, medicine and manufacturing. Furthermore, with our submitted parts, every iGEM team can incorporate these ncAAs into their target proteins.

Regarding our project, two of the ncAAs that are part of our toolbox perform an autocatalytic reaction upon irradiation with ultraviolet light. Therefore, we decided to build our own LED panel that allows us to perform experiments with these non‑canonical amino acids under reproducible irradiation conditions.

Achievements


We established two orthogonal methods for the detection of unnatural base pairs in a target sequence: an Oxford Nanopore sequencing application 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



Colocalization of the RuBisCo and and subcellular compartment (carboxysome) using a fluorescent amino acid


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 for other applications



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


Writing a biosafety report entitled “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”