Difference between revisions of "Team:Bielefeld-CeBiTec"

 
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<div class="container text_header"><h2 style="border-bottom="2px #666633">Evobodies - Molecular Speed Dating</h2> </div>
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<div class="container text">We developed a novel and easy to use system for the generation of binding proteins in <i>E. coli</i> via <i>in vivo</i> directed evolution. Resulting proteins called Evobodies have the potential to bind specifically to target proteins enabling various medical and analytical applications. Great advantages of our low-cost system are the short hands on time and the short generation time.  
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<div class="container text">We developed a novel and easy to use system for the generation of binding proteins in E. coli via in vivo directed evolution. Resulting proteins have the potential to be utilized for therapeutic or diagnostic purposes such as target-mediated drug delivery, immunodetection, or passive immunization. The system is particular useful for quick on-site generation of binders of evolving pathogens such as viruses. Moreover, many applications for basic research are within reach with limited hands on time and at low cost.
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As the starting point of our system, we designed and synthesized a library of sequences encoding binding proteins in E. coli based on Nanobodies as well as Monobodies. These libraries were cloned and are suggested as a new entity of BioBricks. The diversity of the library in E. coli is continuously increased by co-expressing a special DNA-Polymerase inducing plasmid restricted error-prone replication of the binding protein expressing plasmids. Finally, binding proteins with high affinity to the target protein are selected using a bacterial two-hybrid system conferring growth advantage to antibiotics in relation to protein-protein interaction. Ultimately, desired clones are enriched during fermentation either on plates or in continuous culture.  
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Next to cloning the libraries, the selection and the evolution system, we validated the mutation system, defined the selection efficacy of our system and selected binding molecules.
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As the starting point of our system, we designed and synthesized genetic libraries encoding binding proteins based on Nanobodies as well as Monobodies.  
 +
The diversity of the respective library in <i>E. coli</i> is continuously increased by co-expressing a special DNA-Polymerase conferring plasmid restricted error-prone replication of the binding protein expressing plasmids.
 +
Finally, binding proteins with high affinity to the target protein are selected using a bacterial two-hybrid system providing growth advantage to antibiotics in relation to protein-protein interaction strength. Ultimately, desired clones are enriched during fermentation in batch or in continuous culture.
 +
<br>
 +
<br>
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Binding capability of our libraries, efficiency of our selection system and potential of our mutagenesis system were demonstrated. Moreover, library diversity and mutation system characteristics were analyzed in detail by high-throughput sequencing.  
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<h3 class="textHeadline"> <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Project/Mutation">Mutation</a> </h3>
 
<p class="stdText"><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Mutation">Overview</a><br><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Mutation">Results</a></p>
 
<p class="stdText"><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Mutation">Overview</a><br><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Mutation">Results</a></p>
 
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<p class="stdText"><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Project/Selection/Motivation">Overview</a><br><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Selection">Results</a></p>
 
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<h3 class="textHeadline"><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Model"> Modeling</a></h3>
 
<h3 class="textHeadline"><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Model"> Modeling</a></h3>
 
<p class="stdText"><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Model">Overview</a><br><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Modeling">Results</a></p>
 
<p class="stdText"><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Model">Overview</a><br><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Modeling">Results</a></p>
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<td style="border:none; width:20px"><img src="https://static.igem.org/mediawiki/2016/4/48/Bielefeld_CeBiTec_2016_10_18_XXXX_tick.png" class="check" width="40px"></td>
 
<td style="border:none; width:20px"><img src="https://static.igem.org/mediawiki/2016/4/48/Bielefeld_CeBiTec_2016_10_18_XXXX_tick.png" class="check" width="40px"></td>
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<td style="text-align:left; border:none;">Establishment of <i>in vivo</i> <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Project/Mutation" target="_blank">mutagenesis systems</a> for the iGEM community </td>
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<td style="text-align:left; border:none;">
 +
Design and construction of an <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Project/Library/Overview" target="_blank">Evobody library</a> and invention of a new BioBrick class  </td>
 
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<td style="border:none; width:20px"><img src="https://static.igem.org/mediawiki/2016/4/48/Bielefeld_CeBiTec_2016_10_18_XXXX_tick.png" class="check" width="40px"></td>
 +
<td style="text-align:left; border:none;">
 +
Over 100,000 clones per <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Project/Library/Overview" target="_blank">library</a> generated  </td>
 +
</tr>
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 +
</tr>
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<td style="border:none; width:20px"><img src="https://static.igem.org/mediawiki/2016/4/48/Bielefeld_CeBiTec_2016_10_18_XXXX_tick.png" class="check" width="40px"></td>
 +
<td style="text-align:left; border:none;">
 +
High diversity of the plasmid library confirmed by <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Library/Sequencing" target="_blank">high-throughput sequencing </a> </td>
 +
</tr>
 +
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<td style="border:none; width:20px"><img src="https://static.igem.org/mediawiki/2016/4/48/Bielefeld_CeBiTec_2016_10_18_XXXX_tick.png" class="check" width="40px"></td>
 +
<td style="text-align:left; border:none;">
 +
Functionality of the <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Library/Phage" target="_blank">library </a>  was demonstrated by binding to various targets </td>
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<td style="border:none; width:20px"><img src="https://static.igem.org/mediawiki/2016/4/48/Bielefeld_CeBiTec_2016_10_18_XXXX_tick.png" class="check" width="40px"></td>
 
<td style="border:none; width:20px"><img src="https://static.igem.org/mediawiki/2016/4/48/Bielefeld_CeBiTec_2016_10_18_XXXX_tick.png" class="check" width="40px"></td>
−
<td style="text-align:left; border:none;">Design and construction of a <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Project/Library/Overview" target="_blank">library</a> </td>
+
<td style="text-align:left; border:none;">Construction and detailed characterization via high-throughput sequencing of a plasmid-specific <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Mutation" target="_blank"> mutagenesis system </a> </td>
 
</tr>
 
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<td style="border:none; width:20px"><img src="https://static.igem.org/mediawiki/2016/4/48/Bielefeld_CeBiTec_2016_10_18_XXXX_tick.png" class="check" width="40px"></td>
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<td style="text-align:left; border:none;">
 +
Construction and characterization of several parts for <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Mutation/Reversion" target="_blank"> reversion assays </a>  </td>
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</tr>
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<td style="border:none; width:20px"><img src="https://static.igem.org/mediawiki/2016/4/48/Bielefeld_CeBiTec_2016_10_18_XXXX_tick.png" class="check" width="40px"></td>
 +
<td style="text-align:left; border:none;">
 +
Plasmid sequence improvement by <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Mutation/Sequencing" target="_blank"> re-sequencing </a> and  <i>de novo</i> assembly  </td>
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<td style="border:none; width:20px"><img src="https://static.igem.org/mediawiki/2016/4/48/Bielefeld_CeBiTec_2016_10_18_XXXX_tick.png" class="check" width="40px"></td>
 
<td style="border:none; width:20px"><img src="https://static.igem.org/mediawiki/2016/4/48/Bielefeld_CeBiTec_2016_10_18_XXXX_tick.png" class="check" width="40px"></td>
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<td style="text-align:left; border:none;">Establishment of a functional <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Project/Selection" target="_blank">selection system</a> </td>
+
<td style="text-align:left; border:none;">Construction and characterization of a <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Selection" target="_blank"> bacterial two-hybrid selection system </a> </td>
 
</tr>
 
</tr>
 
<tr style="border:none;">
 
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<td style="border:none; width:20px"><img src="https://static.igem.org/mediawiki/2016/4/48/Bielefeld_CeBiTec_2016_10_18_XXXX_tick.png" class="check" width="40px"></td>
 
<td style="border:none; width:20px"><img src="https://static.igem.org/mediawiki/2016/4/48/Bielefeld_CeBiTec_2016_10_18_XXXX_tick.png" class="check" width="40px"></td>
−
<td style="text-align:left; border:none;"> <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Model" target="_blank">Predict</a> how our system work</td>
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<td style="text-align:left; border:none;"> <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Modeling" target="_blank">
 +
Prediction </a> of the outcome of our system</td>
 
</tr>
 
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<tr style="border:none;">
 
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<td style="border:none; width:20px"><img src="https://static.igem.org/mediawiki/2016/4/48/Bielefeld_CeBiTec_2016_10_18_XXXX_tick.png" class="check" width="40px"></td>
 
<td style="border:none; width:20px"><img src="https://static.igem.org/mediawiki/2016/4/48/Bielefeld_CeBiTec_2016_10_18_XXXX_tick.png" class="check" width="40px"></td>
−
<td style="text-align:left; border:none;"> Several <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Human_Practices" target="_blank">human practice projects</a> </td>
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<td style="text-align:left; border:none;"> <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Human_Practices" target="_blank">
 +
Evolution-based human practice projects </a> were perfectly integrated</td>
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<tr style="border:none;">
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<td style="border:none; width:20px"><img src="https://static.igem.org/mediawiki/2016/4/48/Bielefeld_CeBiTec_2016_10_18_XXXX_tick.png" class="check" width="40px"></td>
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<td style="text-align:left; border:none;">
 +
Concept development for  <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Entrepreneurship" target="_blank">industrial upscaling</a> </td>
 
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Latest revision as of 16:24, 1 December 2016




We developed a novel and easy to use system for the generation of binding proteins in E. coli via in vivo directed evolution. Resulting proteins called Evobodies have the potential to bind specifically to target proteins enabling various medical and analytical applications. Great advantages of our low-cost system are the short hands on time and the short generation time.

As the starting point of our system, we designed and synthesized genetic libraries encoding binding proteins based on Nanobodies as well as Monobodies. The diversity of the respective library in E. coli is continuously increased by co-expressing a special DNA-Polymerase conferring plasmid restricted error-prone replication of the binding protein expressing plasmids. Finally, binding proteins with high affinity to the target protein are selected using a bacterial two-hybrid system providing growth advantage to antibiotics in relation to protein-protein interaction strength. Ultimately, desired clones are enriched during fermentation in batch or in continuous culture.

Binding capability of our libraries, efficiency of our selection system and potential of our mutagenesis system were demonstrated. Moreover, library diversity and mutation system characteristics were analyzed in detail by high-throughput sequencing.



Achievements

Design and construction of an Evobody library and invention of a new BioBrick class
Over 100,000 clones per library generated
High diversity of the plasmid library confirmed by high-throughput sequencing
Functionality of the library was demonstrated by binding to various targets
Construction and detailed characterization via high-throughput sequencing of a plasmid-specific mutagenesis system
Construction and characterization of several parts for reversion assays
Plasmid sequence improvement by re-sequencing and de novo assembly
Construction and characterization of a bacterial two-hybrid selection system
Prediction of the outcome of our system
Evolution-based human practice projects were perfectly integrated
Concept development for industrial upscaling