Difference between revisions of "Team:Hannover/Design"

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<h1>Design: Our TALebot-vectors and their assembly</h1>
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<p>In order to generate a TALebot, we used Gibson Assembly to ligate several DNA fragments in a given order.  All linkers, tags, the nuclear localization sequence and the repeats were inserted into the iGEM vector as part of PCR amplification primers with overlapping sequences allowing Gibson Assembly to produce the desired TALE. </p>
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<p>Designing cyclic TALEs allows a regulation of those proteins, because of topological problems. A TALE is always winding itself around the DNA to bind. If the protein is cyclic, this is no longer possible and the TALE-bond is inhibited. This could also be used for appliances concerning drug delivery. If the cyclic bond is irreversible and a protease can cut the protein, the TALE regains full transcriptional activity (Lonzarić, 2016). To prove this statement, we inserted a TEV cleavage site from the tobacco each virus into the vector which enables induced linearization of the protein after expression with ProTEV Plus protease (Promega). </p>
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<img src="TODO"/> TODO Schaubild über Ringschluss und wieder Schneiden mit TEV
  
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<p> Delete this box in order to be evaluated for this medal. See more information at <a href="https://2016.igem.org/Judging/Pages_for_Awards/Instructions"> Instructions for Pages for awards</a>.</p>
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<p>A Strep Tag with two linker sequences by gBlocks was added to the vector to allow a purification of the expressed protein. In this way, we were able to perform anon-column purification with the Strep-tag system developed by IBA. </p>
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<p>Two of our vectors also include an eGFP. Thir green fluorescent protein can be used to detect samples under blue or UV light due to emission of green light. eGFP is an enhanced version of GFP with a higher intensity from Aequorea Victoria. During our experiments, we used eGFP to detect TALEs on the chip spotted with specific DNA. </p>
  
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<p>First, we used BL21 (DE3) cells with a T7 IPTG-inducable promoter to express our protein. But we came across several problems. Our expressed TALE was not circular and a TEV cleavage lead to no results. This is why we did some research and tried purification with DTT as well as expressing our protein in Origami B (DE3) cells. This is a mutation of the original Origami strain enabling the induction of expression with IPTG. In addition and a very important fact for us, Origami strains have mutations in glutathione reductase (gor) and thioredoxin reductase (trxB) which allow disulfide bonding formation in the cytoplasma. DTT and the expression in Origami cells allowed the cyclization of TALEs by stabilizing the peptide bond by disulfide bonds. </p>
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By talking about your design work on this page, there is one medal criterion that you can attempt to meet, and one award that you can apply for. If your team is going for a gold medal by building a functional prototype, you should tell us what you did on this page.
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<p>This is a prize for the team that has developed a synthetic biology product to solve a real world problem in the most elegant way. The students will have considered how well the product addresses the problem versus other potential solutions, how the product integrates or disrupts other products and processes, and how its lifecycle can more broadly impact our lives and environments in positive and negative ways.</p>
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If you are working on art and design as your main project, please join the art and design track. If you are integrating art and design into the core of your main project, please apply for the award by completing this page.
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<p>Teams who want to focus on art and design should be in the art and design special track. If you want to have a sub-project in this area, you should compete for this award.</p>
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<h5>Sponsors</h5>
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  <p><small><strong>Our project would not have been possible without financial support from multiple sponsors and supporters.</strong>
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<img class="sponsorImg" src="TODO" alt="Carl Roth">
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<img class="sponsorImg" src="TODO" alt="IDT">
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<img class="sponsorImg" src="TODO" alt="Leibniz University Hannover ">
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<img class="sponsorImg" src="TODO" alt="Leibniz Universitätsgesellschaft e.V. ">
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<img class="sponsorImg" src="TODO" alt="New England Biolabs ">
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<img class="sponsorImg" src="TODO" alt="Promega">
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<img class="sponsorImg" src="TODO" alt="Sartorius">
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<img class="sponsorImg" src="TODO" alt="SnapGene">
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Revision as of 05:19, 14 October 2016

Design: Our TALebot-vectors and their assembly

In order to generate a TALebot, we used Gibson Assembly to ligate several DNA fragments in a given order. All linkers, tags, the nuclear localization sequence and the repeats were inserted into the iGEM vector as part of PCR amplification primers with overlapping sequences allowing Gibson Assembly to produce the desired TALE.

Designing cyclic TALEs allows a regulation of those proteins, because of topological problems. A TALE is always winding itself around the DNA to bind. If the protein is cyclic, this is no longer possible and the TALE-bond is inhibited. This could also be used for appliances concerning drug delivery. If the cyclic bond is irreversible and a protease can cut the protein, the TALE regains full transcriptional activity (Lonzarić, 2016). To prove this statement, we inserted a TEV cleavage site from the tobacco each virus into the vector which enables induced linearization of the protein after expression with ProTEV Plus protease (Promega).

TODO Schaubild über Ringschluss und wieder Schneiden mit TEV

A Strep Tag with two linker sequences by gBlocks was added to the vector to allow a purification of the expressed protein. In this way, we were able to perform anon-column purification with the Strep-tag system developed by IBA.

Two of our vectors also include an eGFP. Thir green fluorescent protein can be used to detect samples under blue or UV light due to emission of green light. eGFP is an enhanced version of GFP with a higher intensity from Aequorea Victoria. During our experiments, we used eGFP to detect TALEs on the chip spotted with specific DNA.

First, we used BL21 (DE3) cells with a T7 IPTG-inducable promoter to express our protein. But we came across several problems. Our expressed TALE was not circular and a TEV cleavage lead to no results. This is why we did some research and tried purification with DTT as well as expressing our protein in Origami B (DE3) cells. This is a mutation of the original Origami strain enabling the induction of expression with IPTG. In addition and a very important fact for us, Origami strains have mutations in glutathione reductase (gor) and thioredoxin reductase (trxB) which allow disulfide bonding formation in the cytoplasma. DTT and the expression in Origami cells allowed the cyclization of TALEs by stabilizing the peptide bond by disulfide bonds.

TODO
TODO
TODO
TODO
Sponsors

Our project would not have been possible without financial support from multiple sponsors and supporters. Carl Roth IDT Leibniz University Hannover Leibniz Universitätsgesellschaft e.V. New England Biolabs Promega Sartorius SnapGene