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

 
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<img src="https://static.igem.org/mediawiki/2017/6/68/T--Bielefeld-CeBiTec--YKE_toolbox_icon_1.png" width="30%" style="margin:auto; margin-top: 10px; margin-bottom: 20px;">
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Toolbox Overview
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<h2> Toolbox  </h2>
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<h2> Toolbox Overview </h2>
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                     Our toolbox for advanced protein design will consist of seven different tRNA/aminacyl-synthetases (aaRS), each capable of loading a specific non&#x2011;canonical amino acid (ncAA) to the amber tRNA or the less used leucine tRNA to incorporate these ncAAs into all kinds of target proteins. The aaRSs will be provided as parts in the Registry of Standard Biological Parts and hopefully integrated in the distribution, so that further iGEM teams will have the benefit of using our toolbox to improve their projects. The properties we would like to integrate in the toolbox are analysing, photoswitching, labeling, photolysis and fusing (Figure 1).
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                     Our toolbox for advanced protein design consists of eight different tRNA/aminacyl-synthetases (aaRS), each capable of loading a specific non-canonical amino acid (ncAA) to the amber tRNA or the less used leucine tRNA to incorporate these ncAAs into all types of target proteins. The aaRSs are provided as parts in the Registry of Standard Biological Parts, so that further iGEM teams will have the benefit of using our toolbox to improve their projects. The properties we integrated in our toolbox are <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/analysing">Analyzing</a>, <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/photoswitching">Photoswitching</a>, <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/labeling">Labeling</a>, <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/photolysis">Photolysis</a> and <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/fusing">Fusing</a> (Figure 1).
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/6/68/T--Bielefeld-CeBiTec--YKE_toolbox_icon_1.png">
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<p class="figure subtitle"><b>Figure 1: Overview of the different functions provided by specific ncAAs</b><br>We want to integrate plasmids with the sequences of specific synthetases into our toolbox and provide them for the iGEM as well as the synthetic biology community.</p>
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                     <li> <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/analysing">Analysing</a>: Two different ncAAs are incorporated which are labeled with fluorophores in a chemically reaction. With the help of Foerster&nbsp;Resonance&nbsp;Energy&nbsp;Transfer (FRET) the distance between the amino acids could be measured.
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                     <li> <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/analysing">Analyzing</a> (<a target="_blank" href="http://parts.igem.org/Part:BBa_K2201201">BBa_K2201201</a>, <a target="_blank" href="http://parts.igem.org/Part:BBa_K2201202">BBa_K2201202</a>): Two different ncAAs are incorporated which are labeled with fluorophores in a chemically reaction. With the help of Foerster&nbsp;Resonance&nbsp;Energy&nbsp;Transfer (FRET), the distance between the amino acids could be measured, to get information about the conformation.
                     <li> <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/photoswitching">Photoswitching</a>: A photoisomerisable amino acid could be incorporated which changes its conformation when it is irradiated with light of different wavelengths. In one conformation it inhibits the function of the target protein. With this photoswitch, reactions could be switched on and off on the protein level only by light exposure.
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                     <li> <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/photoswitching">Photoswitching</a> (<a target="_blank" href="http://parts.igem.org/Part:BBa_K2201207">BBa_K2201207</a>): A photoisomerisable amino acid could be incorporated which changes its conformation when it is irradiated with light of different wavelengths. This conformation change can be used to inhibit the functionality of the target protein, so that reactions can be switched on and off on the protein level using only light exposure.
                     <li> <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/labeling">Labeling</a>: A fluorescent amino acid could be used to label the target protein <i>in vivo</i>. The advantages compared to fluorescent proteins lay in the smaller size of the fluorescent amino acid.
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                     <li> <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/labeling">Labeling</a> (<a target="_blank" href="http://parts.igem.org/Part:BBa_K2201204">BBa_K2201204</a>): A fluorescent amino acid could be used to label the target protein <i>in vivo</i>. The advantages compared to fluorescent proteins lay in the smaller size of the fluorescent amino acid.
                     <li> <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/photolysis">Photolysis</a>: Photolysis amino acids break the peptide backbone at the position they were incorporated. This could be used to activate or deactivate proteins by cleaving them.
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                     <li> <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/photolysis">Photolysis</a> (<a target="_blank" href="http://parts.igem.org/Part:BBa_K2201200">BBa_K2201200</a>): Photolysis amino acids break the peptide backbone at the position they were incorporated. This cleaving could be used to activate or deactivate proteins.
                     <li> <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/fusing">Fusing</a>: Two ncAAs which could form a specific covalent bond to each other could be used to fuse proteins together or immobilize the target protein not only at the C- or N-termini.  
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                     <li> <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/toolbox/fusing">Fusing</a> (<a target="_blank" href="http://parts.igem.org/Part:BBa_K2201208">BBa_K2201208</a>, <a target="_blank" href="http://parts.igem.org/Part:BBa_K2201302">BBa_K2201302</a>): Two ncAAs which form a specific covalent bond between each other can be used to fuse proteins together or immobilize the target protein, independent of the C- or N-termini.  
 
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                   Our toolbox can be used by transforming the BioBrick of the desired synthetase along with a target protein containing the amber codon. If the co&#x2011;transformation was successful the amber codon will no longer be read as stop signal in the protein synthesis but as a codon to incorporate the specific ncAA and so it will enable the usage of their special property.
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                   Our toolbox can be used by transforming the BioBrick of the desired synthetase along with a target protein containing the amber codon. If the co&#x2011;transformation was successful, the amber codon will no longer be read as a stop signal in the protein synthesis but as a codon to incorporate the specific ncAA and it will enable the usage of their special properties.
                   <br>We hope that our toolkit will be used frequently by future iGEM-teams and help them at their projects. We also wish that they will add new synthetases for other ncAAs and thus expand the possibilities of further applications in protein design.
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                   <br>We hope that our toolbox will be used frequently by future iGEM-teams and help them at their projects. We also wish that they will add new synthetases for other ncAAs and thus expand the possibilities of further applications in protein design.
 
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                 <h2>Two different ways on how to incorporate ncAAs</h2>
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                 <h2>Two Different Ways on how to Incorporate ncAAs</h2>
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                     Generally the incorporation in response to the amber stop codon (TAG) and the less used leucine codon (CTA) limits the number of different non canonical amino acids to two. The incorporation through these codons also inhibits the growth of culture, like described in the translation mechanism. An additional base in the DNA would circumvent these problems and offer 64 new different codons to incorporate different ncAAs like shown in Figure 2. Both ways, the incorporation through codons containing an unnatural base and the incorporation through a less used existing codon could be used to incorporate ncAAs.
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                     Generally, the incorporation in response to the amber stop codon (TAG) and the less used leucine codon (CTA) limits the number of different non-canonical amino acids to two. The incorporation through these codons also inhibits the growth of culture, as described in the <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/translational_system/translation_mechanism">translation mechanism</a>. An additional base in the DNA would circumvent these problems and offers 64 new different codons to incorporate different ncAAs as shown in Figure 2. Both, the incorporation through codons containing an <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Project/unnatural_base_pair/unnatural_base_pairs">unnatural base</a> and the incorporation through a less used existing codon are ways to incorporate ncAAs.
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/6/6c/T--Bielefeld-CeBiTec--YKE_expanded_codesun_1.png">
 
<img class="figure image" src="https://static.igem.org/mediawiki/2017/6/6c/T--Bielefeld-CeBiTec--YKE_expanded_codesun_1.png">
<p class="figure subtitle"><b>Figure 2: Expanded code sun</b><br>Portrayal of the code sun after the incorporation of one new unnatural base to expand the genetic code and create new blank codons (purple) that can be used to evolve aaRS to make many ncAAs available for advanced protein design.</p>
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<p class="figure subtitle"><b>Figure 2: Expanded code sun</b>. Representation of the code sun after the incorporation of one new unnatural base to expand the genetic code and create new blank codons (purple) that can be used to evolve aaRS to enable ncAAs for advanced protein design.</p>
 
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Latest revision as of 03:02, 2 November 2017

Toolbox Overview

Our toolbox for advanced protein design consists of eight different tRNA/aminacyl-synthetases (aaRS), each capable of loading a specific non-canonical amino acid (ncAA) to the amber tRNA or the less used leucine tRNA to incorporate these ncAAs into all types of target proteins. The aaRSs are provided as parts in the Registry of Standard Biological Parts, so that further iGEM teams will have the benefit of using our toolbox to improve their projects. The properties we integrated in our toolbox are Analyzing, Photoswitching, Labeling, Photolysis and Fusing (Figure 1).
  • Analyzing (BBa_K2201201, BBa_K2201202): Two different ncAAs are incorporated which are labeled with fluorophores in a chemically reaction. With the help of Foerster Resonance Energy Transfer (FRET), the distance between the amino acids could be measured, to get information about the conformation.
  • Photoswitching (BBa_K2201207): A photoisomerisable amino acid could be incorporated which changes its conformation when it is irradiated with light of different wavelengths. This conformation change can be used to inhibit the functionality of the target protein, so that reactions can be switched on and off on the protein level using only light exposure.
  • Labeling (BBa_K2201204): A fluorescent amino acid could be used to label the target protein in vivo. The advantages compared to fluorescent proteins lay in the smaller size of the fluorescent amino acid.
  • Photolysis (BBa_K2201200): Photolysis amino acids break the peptide backbone at the position they were incorporated. This cleaving could be used to activate or deactivate proteins.
  • Fusing (BBa_K2201208, BBa_K2201302): Two ncAAs which form a specific covalent bond between each other can be used to fuse proteins together or immobilize the target protein, independent of the C- or N-termini.

Our toolbox can be used by transforming the BioBrick of the desired synthetase along with a target protein containing the amber codon. If the co‑transformation was successful, the amber codon will no longer be read as a stop signal in the protein synthesis but as a codon to incorporate the specific ncAA and it will enable the usage of their special properties.
We hope that our toolbox will be used frequently by future iGEM-teams and help them at their projects. We also wish that they will add new synthetases for other ncAAs and thus expand the possibilities of further applications in protein design.

Two Different Ways on how to Incorporate ncAAs

Generally, the incorporation in response to the amber stop codon (TAG) and the less used leucine codon (CTA) limits the number of different non-canonical amino acids to two. The incorporation through these codons also inhibits the growth of culture, as described in the translation mechanism. An additional base in the DNA would circumvent these problems and offers 64 new different codons to incorporate different ncAAs as shown in Figure 2. Both, the incorporation through codons containing an unnatural base and the incorporation through a less used existing codon are ways to incorporate ncAAs.

Figure 2: Expanded code sun. Representation of the code sun after the incorporation of one new unnatural base to expand the genetic code and create new blank codons (purple) that can be used to evolve aaRS to enable ncAAs for advanced protein design.