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that do not. As we wanted our whole system to work <i>in vivo</i> utilizing directed evolution we settled for three different | that do not. As we wanted our whole system to work <i>in vivo</i> utilizing directed evolution we settled for three different | ||
approaches to screen for functional Evobodies: | approaches to screen for functional Evobodies: | ||
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<div class="container text_header"><h3>Overview</h3></div> | <div class="container text_header"><h3>Overview</h3></div> | ||
− | + | <center><img src="https://static.igem.org/mediawiki/parts/0/05/Bielefeld_CeBiTec_2016_10_13_SEL_BTH_Overview.png" usemap="#overview" width="90%" /></center> | |
The <a href="#">bacterial two-hybrid system</a>, our main approach, consists of two fusion proteins and a reporter gene that grants selective advantage. One | The <a href="#">bacterial two-hybrid system</a>, our main approach, consists of two fusion proteins and a reporter gene that grants selective advantage. One | ||
fusion protein is the target protein fused to a DNA-binding domain while the other comprises the Evobody consisting of a binding protein with a RNA polymerase subunit. The | fusion protein is the target protein fused to a DNA-binding domain while the other comprises the Evobody consisting of a binding protein with a RNA polymerase subunit. The | ||
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which then could be used for production. | which then could be used for production. | ||
<br><br> | <br><br> | ||
− | <img align="left" class="img-responsive" src=" | + | <img align="left" class="img-responsive" src="https://static.igem.org/mediawiki/2016/3/3c/Bielefeld_CeBiTec_2016_10_19_SEL_splitbla.png" width=40% /> |
The <a href="#">split-protein</a> approach is also based on beta-lactamase as a resistance to ampicillin. The general idea is, to split a reporter protein | The <a href="#">split-protein</a> approach is also based on beta-lactamase as a resistance to ampicillin. The general idea is, to split a reporter protein | ||
into two non-functional subunits. Then, a translational fusion between one half of the split-protein respectively and one of the two allegedly binding proteins | into two non-functional subunits. Then, a translational fusion between one half of the split-protein respectively and one of the two allegedly binding proteins | ||
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functional beta-lactamase. | functional beta-lactamase. | ||
<br><br> | <br><br> | ||
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The <a href="#">TAT-hitchhiker selection</a> works on a similar premise as the split-protein. But in this case the beta-lactamase is not divided into two subunits | The <a href="#">TAT-hitchhiker selection</a> works on a similar premise as the split-protein. But in this case the beta-lactamase is not divided into two subunits | ||
but instead its natural export signals are removed. this modified beta-lactamase forms a translational unit with the target protein whilst a signal peptide | but instead its natural export signals are removed. this modified beta-lactamase forms a translational unit with the target protein whilst a signal peptide | ||
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In summary, we wanted a selection system to screen for Evobodies with high affinity to the target protein while being functional <i>in vivo</i>. We chose three approaches, the | In summary, we wanted a selection system to screen for Evobodies with high affinity to the target protein while being functional <i>in vivo</i>. We chose three approaches, the | ||
<a href="#">bacterial two-hybrid system</a>, the <a href="#">split-protein</a> approach and the <a href="#">TAT-hitchhiker selection</a>. | <a href="#">bacterial two-hybrid system</a>, the <a href="#">split-protein</a> approach and the <a href="#">TAT-hitchhiker selection</a>. | ||
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</div> | </div> | ||
+ | <div class="container text_header"><h3>References</h3></div> | ||
</div> | </div> | ||
Revision as of 22:15, 19 October 2016
Selection
Motivation
Selection is a key mechanism of evolution like Darwin defined it. Biodiversity and life as we know it in all its complexity is scientificly not explicable without it. We wanted to design a system able to generate an Evobody that can gain affinity towards any target protein it is confronted with. So what better way is there to design our system than copying one of evolutions core principles and letting the bacteria with the best Evobody emerge by itself?
Having a library of Evobodies as well as a mutation system is a great way to guarantee a high variety of binding proteins. However, there is still the need of a screening method to separate Evobodies that show affinity to the target protein from those that do not. As we wanted our whole system to work in vivo utilizing directed evolution we settled for three different approaches to screen for functional Evobodies:
Having a library of Evobodies as well as a mutation system is a great way to guarantee a high variety of binding proteins. However, there is still the need of a screening method to separate Evobodies that show affinity to the target protein from those that do not. As we wanted our whole system to work in vivo utilizing directed evolution we settled for three different approaches to screen for functional Evobodies:
Overview
![](https://static.igem.org/mediawiki/parts/0/05/Bielefeld_CeBiTec_2016_10_13_SEL_BTH_Overview.png)
![](https://static.igem.org/mediawiki/2016/3/3c/Bielefeld_CeBiTec_2016_10_19_SEL_splitbla.png)
The TAT-hitchhiker selection works on a similar premise as the split-protein. But in this case the beta-lactamase is not divided into two subunits but instead its natural export signals are removed. this modified beta-lactamase forms a translational unit with the target protein whilst a signal peptide for the Tat (Twin-Arginine Translocation) pathway is attached to the Evobody. Only in case of a funtionally binding Evobody to the target protein is the beta-lactamase exported into the periplasm where it can counteract the ampicillin. Since the Tat pathway only exports correctly folded proteins, this approach also provides an option to check if the proteins are expressed as we hope they are.
In summary, we wanted a selection system to screen for Evobodies with high affinity to the target protein while being functional in vivo. We chose three approaches, the bacterial two-hybrid system, the split-protein approach and the TAT-hitchhiker selection.