Difference between revisions of "Team:Bielefeld-CeBiTec"

 
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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.
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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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As the starting point of our system, we designed and synthesized a library of a new entity of BioBricks encoding binding proteins in <i>E. coli</i> based on Nanobodies as well as Monobodies. The diversity of the 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.
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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.  
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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.
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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.
 
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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.
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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>
 
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<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>
 
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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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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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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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Over 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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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>
 
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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="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>
 
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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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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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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