Difference between revisions of "Team:Bielefeld-CeBiTec"

 
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<h2> Welcome to iGEM Bielefeld-CeBiTec 2016! </h2>
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<p>Our team is looking forward to start the iGEM season! </p>
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<h5> Abstract: </h5>
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<p align="center"><h3>Directed evolution of binding proteins</h3><br></p>
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<p align="center">Carsten Hain, Niklas Hoffmann, Judith Kampa, Marius Schöller, Mikail Sahin, Pascal Schmidt, Marten Linder, Cassandra Königs, Bianca Frommer, Fabian Roeloffs and Sebastian Perez Knoche</p><br>
 
  
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<p align="center">iGEM Bielefeld-CeBiTec, Bielefeld University, Germany </p><br><hr>
 
  
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The impact of antibodies in modern medicine is on a permanent rise but the cost and time factor as well as the immunization of animals, which die during the harvesting process, are still problematic. Therefore we want to establish an alternative using antibody-like binding proteins that are generated <i>in vivo</i> in <i>E. coli</i>. Profiting of the short generation cycle and the exponential growth of bacteria, we want to generate binding proteins in a much shorter period of time while also being more cost efficient. Furthermore, no animals have to suffer in the process by immunization.
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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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Our goal is to develop binding proteins in <i>E. coli</i> in a process of directed evolution that can subsequently be utilized in diagnostic techniques and target-mediated drug delivery against pathogens. Due to the ability of our system to quickly adapt to a certain target protein under evolutionary pressure it is especially useful in concern of quickly evolving and newly arising viral pathogens. The concept of our system subdivides into the following aspects: At first, we create a randomized library of binding protein sequences in bacteria to form the starting point of our project. As scaffolds for our binding proteins, we settled on both antibody mimetics (monobodies) and natural antibody fragments (nanobodies). In the next step, we use a two plasmid system in combination with a mutant of the polymerase I which leads to a higher mutation rate in the coding regions of our binding proteins, so that there is a chance for the binding proteins to adapt to the target proteins. Finally, we isolate the strains that produce the binding proteins with the highest affinity to the target protein by using an <i>in vivo</i> selection mechanisms: Mediated by the binding of our protein and the target, the cell in concern is granted a selective advantage directly increasing its evolutionary fitness. </p>
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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 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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>Library</a></h3>
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>Overview</a> <br><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Results/Library/Overview">Results</a></p>
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<h3 class="textHeadline"><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Project/Selection/Motivation">Selection</a></h3>
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<h3 class="textHeadline"><a href="https://2016.igem.org/Team:Bielefeld-CeBiTec/Model"> Modeling</a></h3>
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<div class="container text_header"><h3>Achievements</h3></div>
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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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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>
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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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<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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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="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>
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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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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  <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>
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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>
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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>
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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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<td style="text-align:left; border:none;">
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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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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