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

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                     <li> Fusing: 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.  
 
                     <li> Fusing: 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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                   Our toolkit 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 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.
 
                   Our toolkit 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 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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                   <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.<br>
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                   <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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Revision as of 00:16, 27 August 2017

Overview

Toolkit

Our toolkit for advanced protein design will consist of seven 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 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 toolkit to improve their projects. The properties we would like to integrate in the toolkit are analysing, photoswitching, labeling, photolysis and fusing (figure 1).

Figure 1: Overview of the different functions provided by specific ncAAs
We want to integrate plasmids with the sequences of specific synthetases into our toolkit and provide them for the iGEM as well as the synthetic biology community.

  • Analysing: 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.
  • Photoswitching: 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.
  • Labeling: 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: 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.
  • Fusing: 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.

Our toolkit 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 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.
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.

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, 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.

Figure 2: Expanded code sun
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.