Difference between revisions of "Team:Bielefeld-CeBiTec"

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<img class="picture" src="https://static.igem.org/mediawiki/2016/0/03/Bielefeld_CeBiTec_2016_10_13_X_Evobodies_animation.gif" >
 
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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 specific to target proteins enabling varies medical and analytical applications. Big advantages of our low-cost system are the short hands on time and the short generation time.
 
<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 specific to target proteins enabling varies medical and analytical applications. Big advantages of our low-cost system are the short hands on time and the short generation time.
 
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Binding capacities 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.
 
Binding capacities 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/Mutation">Mutation</a> </h3>
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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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<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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Design and construction of a <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>
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DeOver 100,000 clones per <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Project/Library/Overview" target="_blank">library</a> generated  </td>
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High diversity confirmed by <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Library/Sequencing" target="_blank">high-throughput sequencing </a> </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>
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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>
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<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>
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<td style="text-align:left; border:none;">Construction, detailed characterization via high-throughput sequencing and submission of a plasmid-specific <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Mutation" target="_blank"> mutagenesis systems </a> </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>
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<td style="text-align:left; border:none;">
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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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<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;">
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Plasmid sequence improvement by <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Mutation/Sequencing" target="_blank"> re-sequencing </a> and <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Mutation/Assembly" target="_blank"> <i>de novo</i> assembly  reversion assays</a>  </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>
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<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>
 
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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;"> <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">
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Prediction </a> of the outcome of our system</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;"> 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">
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Evolution-based human practice projects </a> were perfectly integrated</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>
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Concept development for  <a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Entrepreneurship" target="_blank">industrial upscaling</a> </td>
 
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Revision as of 02:53, 20 October 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 specific to target proteins enabling varies medical and analytical applications. Big 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 a library of a new entity of BioBricks encoding binding proteins in E. coli based on Nanobodies as well as Monobodies. The diversity of the 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 capacities 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 a Evobody library and invention of a new BioBrick class
DeOver 100,000 clones per library generated
High diversity confirmed by high-throughput sequencing
Functionality of the library was demonstrated by binding to various targets
Construction, detailed characterization via high-throughput sequencing and submission of a plasmid-specific mutagenesis systems
Construction and characterization of several parts for reversion assays
Plasmid sequence improvement by re-sequencing and de novo assembly reversion assays
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