Difference between revisions of "Team:Hannover/Results"

 
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           <a data-toggle="collapse" href="#collapseMenu"><strong>Projects</strong></a>
+
           <a data-toggle="collapse" href="#collapseMenu"><strong>Projects<span class="glyphicon glyphicon-chevron-down" aria-hidden="true"></span></strong></a>
 
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         <h4 class="panel-title">
           <a data-toggle="collapse" href="#collapseTALebots"><strong>TALebots</strong></a>
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           <a data-toggle="collapse" href="#collapseTALebots"><strong>TALebots <span class="glyphicon glyphicon-chevron-down" aria-hidden="true"></span></strong></a>
 
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         <ul class="list-group">
 
         <ul class="list-group">
           <li>Description</li>
+
           <li><a href="https://2016.igem.org/Team:Hannover/Description">Description</a></li>
  <li>Design</li>
+
  <li><a href="https://2016.igem.org/Team:Hannover/Design">Design</a></li>
  <li>Modelling</li>
+
  <!--li><a href="???">Modelling</a></li-->
  <li>Proof</li>
+
  <li><a href="https://2016.igem.org/Team:Hannover/Demonstrate">Demonstrate</a></li>
  <li>Demonstrate</li>
+
  <li><a href="https://2016.igem.org/Team:Hannover/Proof">Proof</a></li>
  <li>Results</li>
+
  <li><a href="https://2016.igem.org/Team:Hannover/Results">Results</a></li>
 +
  <li><a href="https://2016.igem.org/Team:Hannover/Reference">Reference</a></li>
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           <a data-toggle="collapse" href="#collapseSW"><strong>Software Tools</strong></a>
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           <a data-toggle="collapse" href="#collapseSW"><strong>Software Tools <span class="glyphicon glyphicon-chevron-down" aria-hidden="true"></span></strong></a>
 
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           <li>Description</li>
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           <li><a href="https://2016.igem.org/Team:Hannover/Software#description">Description</a></li>
  <li>Design</li>
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  <li><a href="https://2016.igem.org/Team:Hannover/Software#phabricator">Phabricator</a></li>
  <li>Modelling</li>
+
  <li><a href="https://2016.igem.org/Team:Hannover/Software#cyberprinter">CyberPrinter</a></li>
 +
  <li><a href="https://2016.igem.org/Team:Hannover/Software#talsetter">Modeling: TALsetter</a></li>
 +
  <!--li><a href="TODO">Design</a></li-->
 +
  <!--li>Modelling</li-->
 
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           <a data-toggle="collapse" href="#collapseInterLab"><strong>InterLab Study</strong></a>
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           <a href="https://2016.igem.org/Wiki/Team:Hannover/InterLab"><strong>InterLab Study</strong></a>
 
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        <ul class="list-group">
 
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<p>The first part of the results section deals with the cloning of TALebots by using Golden Gate Cloning. After cloning, we tested our vectors with a control digest and a colony PCR. This is how we proved that the vectors used for the following experiments contain the desired parts and that the assembly was successful.</p>
 
<p>The first part of the results section deals with the cloning of TALebots by using Golden Gate Cloning. After cloning, we tested our vectors with a control digest and a colony PCR. This is how we proved that the vectors used for the following experiments contain the desired parts and that the assembly was successful.</p>
 
<figure style="text-align:center;">
 
<figure style="text-align:center;">
<img src="https://static.igem.org/mediawiki/2016/2/24/T--Hannover--ControlDigest.png" class="center-block" height="350px;"/>
+
<img id="fig11" src="https://static.igem.org/mediawiki/2016/2/24/T--Hannover--ControlDigest.png" class="center-block" height="300px;"/>
 
<figcaption style="text-align:center;"><small>Figure 11: Control digests of our assembled vectors</small></figcaption>
 
<figcaption style="text-align:center;"><small>Figure 11: Control digests of our assembled vectors</small></figcaption>
 
</figure>
 
</figure>
<p>The control digest was done using XbaI and PstI. With this digest, we proved that all the inserts are in the vector. We used a 1% agarose gel and ran it at 100 V for 27 min. The 1 kb DNA ladder from Thermo scientific was used as marker.</p>
+
<p>The control digest was done using XbaI and PstI. With this digest, we proved that all the inserts are in the vector. </p>
<p>Digests of the different samples A, B, C and D should result in two bands of 2044 bp and 3352 bp respectively. As it can be seen in Figure 11, most clones resulted in the expected band pattern.</p>
+
<p>Digests of the different samples A, B, C and D should result in two bands of 2044 bp and 3352 bp respectively. As it can be seen in Figure <a href="#11">11</a>, most clones resulted in the expected band pattern.</p>
<p>For the samples 2.1.1, 3.1.1, 3.2.1. 4.1.1 and 1.1.1, we expected bands with a size around 3695 bp and 2044 bp. The results met our expectations.</p>
+
<p>For the samples 2.1.1, 3.1.1, 3.2.1. 4.1.1 and 1.1.1, we expected bands with a size around 3695 bp and 2044 bp. The results met our expectations. </p>
<p>These two vectors were used for all following experiments, for example the stability tests and the TEV digest with the TEV protease.</p>
+
<p>These two vectors were used for all following experiments, for example, the stability tests and the TEV digest with the TEV protease.</p>
 
<figure style="text-align:center;">
 
<figure style="text-align:center;">
<img src="https://static.igem.org/mediawiki/2016/b/b6/T--Hannover--Kontroll-PCR.jpg" class="center-block" height="350px;"/>
+
<img id="fig12" src="https://static.igem.org/mediawiki/2016/b/b6/T--Hannover--Kontroll-PCR.jpg" class="center-block" height="350px;"/>
 
<figcaption style="text-align:center;"><small>Figure 12: 1% Agarose gel of a control PCR</small></figcaption>
 
<figcaption style="text-align:center;"><small>Figure 12: 1% Agarose gel of a control PCR</small></figcaption>
 
</figure>
 
</figure>
<p>The colony-PCR of sample Ax7L-DS, Hax3-2xNG, Hax3-2xNN and a water control was done with two primers. These primers are used to prove that the sample contains TALE+GFP. It is indicated by a band at 717 bp on an agarose gel. The size marker used for the 1% agarose gel is 100 bp Plus DNA ladder from Thermo Scientific. The gel ran for 27 minutes at 100 V.</p>
+
<p>The colony-PCR of sample Ax7L-DS, Hax3-2xNG, Hax3-2xNN and a water control was done with two primers. These primers are used to prove that the sample contains TALE+GFP. It is indicated by a band at 717 bp on an agarose gel. </p>
<p>For sample Ax7L-DS and the water control, there was no GFP band expected and there is also no band on the gel. For sample Hax3-2xNG and Hax3-2xNN, we expected bans however only Hax3-2xNG portrayed one at the right place. The only band for sample Hax3-2xNN was bigger than 1000 bp.</p>
+
<p>Colony PCR of both samples should result in a band of about 700 bp since this is the size of the <i>gfp</i>-gene. As it is shown in figure <a href="#fig12">12</a>, only sample Hax3-2xNG reveals the expected size whereas sample Hax3-2xNN is consisting of about 1 kB is much larger than expected. </p>
<p>These results show that the assembly of GFP of sample Hax3-2xNG was successful.</p>
+
<p>These results show that the assembly of GFP of sample Hax3-2xNG was successful. </p>
 +
<p>In order to be sure that the assembly was successful, we sequenced A7L-DS. You can find the results <a href="https://static.igem.org/mediawiki/2016/6/69/T--Hannover--Sequenzierungsergebnis111.png">here</a>. All used vectors were assembled correctly and used for the following experiments. </p>
  
<h3>Purification of the protein using a Strep-Column</h3>
+
<h3>Protein expression</h3>
<p>After purifying our expressed protein samples with the gravity flow Strep-Tactin Sepharose column (IBA) using the Strep-Tag, we checked the results with several SDS-PAGEs and immunostain.</p>
+
<p>We applied various conditions for induction of the expression of TALebots in <i>E. coli</i> BL21. Figure <a href="#fig13">13</a> shows such a crude extract of Hax3-2xNG as an example (sample “Raw”). </p>
<p>The next figure shows the results of another purification via a Strep-Tactin Sepharose column. We used the samples of the vector iGEM_02 Ax7L-DS after Expression in BL21 DE3 cells.</p>
+
<p>In the membrane, you see the elution steps from the purification, the precolumn sample and the direct flow-through sample from the column. The precolumn sample and the direct flow-through sample show a protein mix as we have expected.</p>
+
 
<figure style="text-align:center;">
 
<figure style="text-align:center;">
<img src="https://static.igem.org/mediawiki/2016/f/f9/T--Hannover--GelAufgereinigtTALE.jpg" class="center-block" height="350px;"/>
+
<img id="fig13" src="https://static.igem.org/mediawiki/2016/f/f0/T--Hannover--Gel2.jpg" class="center-block" />
<figcaption style="text-align:center;"><small>Figure 13: SDS-Page with the purified samples</small></figcaption>
+
<figcaption style="text-align:center;"><small>Figure 13: SDS-PAGE with the purified samples</small></figcaption>
 
</figure>
 
</figure>
<p>In figure 14 and 15, we tried to confirm the existence of our protein in various samples.</p>
+
<p>Expression of TALebots could be confirmed by an immunostain using an anti-Strep tag antibody (IgG1 Anti – Strep – tagII monoclonal antibody by IBA). After the first promising results, purifications using Strep tactin columns have been performed. </p>
<p>As shown in the figure, the purification was successful. We detected bands using a Strep antibody. In addition, most of the protein was detected in sample E6. This is the sample that we used for the following experiments.</p>
+
 
<div class="row">
+
<h3>Purification of the protein using Strep Tactin and StrepXT Tactin columns</h3>
<div class="col-sm-6">
+
<p>Following, we focused our efforts on the optimization of the purification protocol. We tried different Strep-Tag columns, changed the elution conditions and used DTT during the purification to enable disulfide bond formation. </p>
 +
<p>After purifying our expressed protein samples with the gravity flow Strep-Tactin Sepharose column (IBA) using the Strep-Tag, we checked the results with several coomassie stains and immunostains. As a Strep antibody we used: IgG1 Anti – Strep – tagII monoclonal antibody by Iba Life Sciences. The second antibody was: ECL – Anti-mouse IgG Horseradish Peroxidase-linked whole antibody by GE. </p>
 +
<p>Whereas the immunostain showed bonds and verified the TALebot, we could not detect clear protein bands using coomassie stain. </p>
 +
<p>Finally, we were able to highly purify the TALebot, as it is shown in Figure <a href="#fig14">14</a>. Samples applied to the gel in Figure <a href="#fig14">14</a> resulted from BL21 cells transformed with the vector containing TALE Ax7L-DS. </p>
 +
 
 
<figure style="text-align:center;">
 
<figure style="text-align:center;">
<img src="https://static.igem.org/mediawiki/2016/7/7c/T--Hannover--Gel1.jpg" class="center-block" height="350px;"/>
+
<img id="fig14" src="https://static.igem.org/mediawiki/2016/f/f9/T--Hannover--GelAufgereinigtTALE.jpg" class="center-block" />
<figcaption style="text-align:center;"><small>Figure 14: SDS-Page with samples from the purification</small></figcaption>
+
<figcaption style="text-align:center;"><small>Figure 14: SDS-PAGE with samples from the purification</small></figcaption>
 
</figure>
 
</figure>
</div>
+
 
<div class="col-sm-6">
+
<p>In figure <a href="#15">15</a>, we tried to confirm the existence of our protein in various samples. </p>
 +
<p>As shown in the figure, the purification was successful. We detected bands using a Strep antibody. In addition, most of the protein was detected in sample E6. This is the sample that we used for the following experiments. </p>
 +
<p>E6 provides also the first double band that was detected on an immunostain during our experiments. Double bands can show that the protein is circular <a href="Reference"> (Heidelberg, 2014)</a>. Therefore, the purification of Hax3-2xNN successfully provided us with circular TALebots. </p>
 
<figure style="text-align:center;">
 
<figure style="text-align:center;">
<img src="https://static.igem.org/mediawiki/2016/f/f0/T--Hannover--Gel2.jpg" class="center-block" height="350px;"/>
+
<img id="fig15" src="https://static.igem.org/mediawiki/2016/7/7c/T--Hannover--Gel1.jpg" class="center-block" />
 
<figcaption style="text-align:center;"><small>Figure 15: SDS-PAGE with samples from the purification </small></figcaption>
 
<figcaption style="text-align:center;"><small>Figure 15: SDS-PAGE with samples from the purification </small></figcaption>
 
</figure>
 
</figure>
</div>
+
 
</div>
+
<h3>Conclusions</h3>
<p>In Figure 15, we also checked samples from another TEV digest of TALE Ax7L-DS.</p>
+
<p>After finalizing our experiments and describing our results, we want to draw a final conclusion for everybody to get an overview of our project. </p>
<p>As shown above, there are strong bands after 0h, 1h, 2h, and 4h. For more information, take a look at the description of a TEV digest.</p>
+
<p>Originally, we planned to circularize the TALE protein to produce a TALebot. This synthetic protein should result in much higher stability, similar to the ring of fire proteins, presented by the Heidelberg iGEM-team in 2014. The resulting conformation should be tested by several experiments including a TEV digest followed by immune-detection of the resulting fragments using two different tags, which are located on both ends of the linearized protein. </p>
<h3>What are our final results?</h3>
+
<p>The assembly of our TALebot via Golden Gate cloning could be verified by restriction digest analysis and sequencing. You can find the results <a href="https://static.igem.org/mediawiki/2016/6/69/T--Hannover--Sequenzierungsergebnis111.png"> here</a>. </p>
<p>After finalizing our experiments and describing our SDS gels, we want to draw a final conclusion for everybody to get an overview of our project.</p>
+
<p>We began the heterologous production of TALebots in <i>E. coli</i> BL21 (DE3) and Origami 2(DE3) cells with an IPTG-inducible T7 promoter. By changing many parameters of our isolation protocol, e.g. bacterial strain, induction time, compounds of extraction buffer, type of Strep columns etc. we were able to obtain highly purified TALebot protein. Nevertheless, the yield was much too low for most experiments we have planned before. Furthermore, the <a href="#fig15">circularization of the protein could not be reproduced </a>. </p>
<p>Originally, we planned to circularize the TALE protein and test its stability and conformation with several experiments including a TEV digest.</p>
+
<p>We tried to circumvent a possible malfunction of <i>in vivo</i> circularization by adding DTT as an initiator for circularization at different steps of the purification protocol. But these attempts have also not been successful. Therefore, we conclude, that for the expression of TALebot <i>E. coli</i> is not well suited. In fact, during the initial experiments, a publication used circular TALEN for the first time (Lonzaric et al., 2016), but these have been produced in human cell culture, which is not permitted by iGEM due to safety reasons. </p>
<p>After the assembly of our TALE and the Golden Gate cloning, we verified the results with several agarose gels and sequencing. You can find the results <a href="TODO">here </a> <!--TODO (Link zu Sequenzierungsdateien). --> Our cloning was successful.</p>
+
<p>In parallel to the experiments listed above we already started the microarray experiments as a proof of principle for the medical or industrial use of TALebot. These experiments were hindered by several problems in DNA synthesis of Eurofins Company, therefore, we can present these results in our presentation at the Jamboree only.</p>
<p>We produced the protein in E. coli BL21 (DE3) and Origami 2(DE3) cells with an IPTG-inducible T7 promoter. Unfortunately, the purification of the protein was not as successful as expected. The gels showed only blurry bands and we detected them using immunostain. This leads to the conclusion that the expression was not as efficient as expected and protein concentration was not high enough. We did use E. coli cells like the Heidelberg team did in 2014, but unfortunately, it did not work.</p>
+
 
<p>But nevertheless, we continued our work and started stability tests and TEV digests.</p>
+
<h4>Concluding remarks:<h4>
<p>The TEV digests showed some results so we could detect protein. However, we detected multiple linear TALE proteins instead of circular proteins.</p>
+
<p>Our experiments reveal that <i>E. coli</i> is not well suited for the heterologous production of circular TALebots. The most promising solution would be the production in cells from higher eukaryotes, like human embryonic kidney cells (HEK, (Lonzaric et al., 2016). But these experiments cannot be part of the iGEM contest due to safety reasons. </p>
<p>Our microarray experiments are not yet finished. We will continue working on this part of our project until the Jamboree in Boston and show you the results.</p>
+
<p> In addition, using other methods to purify and detect the protein e.g. His-Tag or magnetic beads is an option. This would increase the purification rate. Sadly, we did not have enough time to conduct those experiments. </p>
<p>After finishing our experiments, we researched alternative methods that could be used to circularize the TALEs effectively. Some possible changes include:</p>
+
<p>In the future, TALebots will be produced and perform their job – to simplify the use of TALEs for genome editing. </p>
<ul>
+
 
<li>Expressing the protein in different cell strains (e.g. human embryonic kidney (HEK) cells). Other papers show that the circularization is possible if human cells are used as an expression system (Lonzaric & al, 2016). Unfortunately, we could not try this method because working with those cells would require another lab and working with human cells is not possible during the competition.</li>
+
<li>Using other methods to purify and detect the protein e.g. His-Tag or magnetic beads. This would be an option to increase the purification rate. Sadly, we did not have enough time to conduct this experiments.</li>
+
</ul>
+
 
</div>
 
</div>
 
     <div class="col-sm-2 sidenav">
 
     <div class="col-sm-2 sidenav">

Latest revision as of 23:45, 19 October 2016

Results

Assembly of TALebots

The first part of the results section deals with the cloning of TALebots by using Golden Gate Cloning. After cloning, we tested our vectors with a control digest and a colony PCR. This is how we proved that the vectors used for the following experiments contain the desired parts and that the assembly was successful.

Figure 11: Control digests of our assembled vectors

The control digest was done using XbaI and PstI. With this digest, we proved that all the inserts are in the vector.

Digests of the different samples A, B, C and D should result in two bands of 2044 bp and 3352 bp respectively. As it can be seen in Figure 11, most clones resulted in the expected band pattern.

For the samples 2.1.1, 3.1.1, 3.2.1. 4.1.1 and 1.1.1, we expected bands with a size around 3695 bp and 2044 bp. The results met our expectations.

These two vectors were used for all following experiments, for example, the stability tests and the TEV digest with the TEV protease.

Figure 12: 1% Agarose gel of a control PCR

The colony-PCR of sample Ax7L-DS, Hax3-2xNG, Hax3-2xNN and a water control was done with two primers. These primers are used to prove that the sample contains TALE+GFP. It is indicated by a band at 717 bp on an agarose gel.

Colony PCR of both samples should result in a band of about 700 bp since this is the size of the gfp-gene. As it is shown in figure 12, only sample Hax3-2xNG reveals the expected size whereas sample Hax3-2xNN is consisting of about 1 kB is much larger than expected.

These results show that the assembly of GFP of sample Hax3-2xNG was successful.

In order to be sure that the assembly was successful, we sequenced A7L-DS. You can find the results here. All used vectors were assembled correctly and used for the following experiments.

Protein expression

We applied various conditions for induction of the expression of TALebots in E. coli BL21. Figure 13 shows such a crude extract of Hax3-2xNG as an example (sample “Raw”).

Figure 13: SDS-PAGE with the purified samples

Expression of TALebots could be confirmed by an immunostain using an anti-Strep tag antibody (IgG1 Anti – Strep – tagII monoclonal antibody by IBA). After the first promising results, purifications using Strep tactin columns have been performed.

Purification of the protein using Strep Tactin and StrepXT Tactin columns

Following, we focused our efforts on the optimization of the purification protocol. We tried different Strep-Tag columns, changed the elution conditions and used DTT during the purification to enable disulfide bond formation.

After purifying our expressed protein samples with the gravity flow Strep-Tactin Sepharose column (IBA) using the Strep-Tag, we checked the results with several coomassie stains and immunostains. As a Strep antibody we used: IgG1 Anti – Strep – tagII monoclonal antibody by Iba Life Sciences. The second antibody was: ECL – Anti-mouse IgG Horseradish Peroxidase-linked whole antibody by GE.

Whereas the immunostain showed bonds and verified the TALebot, we could not detect clear protein bands using coomassie stain.

Finally, we were able to highly purify the TALebot, as it is shown in Figure 14. Samples applied to the gel in Figure 14 resulted from BL21 cells transformed with the vector containing TALE Ax7L-DS.

Figure 14: SDS-PAGE with samples from the purification

In figure 15, we tried to confirm the existence of our protein in various samples.

As shown in the figure, the purification was successful. We detected bands using a Strep antibody. In addition, most of the protein was detected in sample E6. This is the sample that we used for the following experiments.

E6 provides also the first double band that was detected on an immunostain during our experiments. Double bands can show that the protein is circular (Heidelberg, 2014). Therefore, the purification of Hax3-2xNN successfully provided us with circular TALebots.

Figure 15: SDS-PAGE with samples from the purification

Conclusions

After finalizing our experiments and describing our results, we want to draw a final conclusion for everybody to get an overview of our project.

Originally, we planned to circularize the TALE protein to produce a TALebot. This synthetic protein should result in much higher stability, similar to the ring of fire proteins, presented by the Heidelberg iGEM-team in 2014. The resulting conformation should be tested by several experiments including a TEV digest followed by immune-detection of the resulting fragments using two different tags, which are located on both ends of the linearized protein.

The assembly of our TALebot via Golden Gate cloning could be verified by restriction digest analysis and sequencing. You can find the results here.

We began the heterologous production of TALebots in E. coli BL21 (DE3) and Origami 2(DE3) cells with an IPTG-inducible T7 promoter. By changing many parameters of our isolation protocol, e.g. bacterial strain, induction time, compounds of extraction buffer, type of Strep columns etc. we were able to obtain highly purified TALebot protein. Nevertheless, the yield was much too low for most experiments we have planned before. Furthermore, the circularization of the protein could not be reproduced .

We tried to circumvent a possible malfunction of in vivo circularization by adding DTT as an initiator for circularization at different steps of the purification protocol. But these attempts have also not been successful. Therefore, we conclude, that for the expression of TALebot E. coli is not well suited. In fact, during the initial experiments, a publication used circular TALEN for the first time (Lonzaric et al., 2016), but these have been produced in human cell culture, which is not permitted by iGEM due to safety reasons.

In parallel to the experiments listed above we already started the microarray experiments as a proof of principle for the medical or industrial use of TALebot. These experiments were hindered by several problems in DNA synthesis of Eurofins Company, therefore, we can present these results in our presentation at the Jamboree only.

Concluding remarks:

Our experiments reveal that E. coli is not well suited for the heterologous production of circular TALebots. The most promising solution would be the production in cells from higher eukaryotes, like human embryonic kidney cells (HEK, (Lonzaric et al., 2016). But these experiments cannot be part of the iGEM contest due to safety reasons.

In addition, using other methods to purify and detect the protein e.g. His-Tag or magnetic beads is an option. This would increase the purification rate. Sadly, we did not have enough time to conduct those experiments.

In the future, TALebots will be produced and perform their job – to simplify the use of TALEs for genome editing.

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