Team:Hannover/Results
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- Description
- Design
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- Results
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.
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.
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.
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.
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.
These results show that the assembly of GFP of sample Hax3-2xNG was successful.
Purification of the protein using a Strep-Column
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.
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.
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.
In figure 14 and 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.
In Figure 15, we also checked samples from another TEV digest of TALE Ax7L-DS.
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.
What are our final results?
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.
Originally, we planned to circularize the TALE protein and test its stability and conformation with several experiments including a TEV digest.
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 here Our cloning was successful.
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.
But nevertheless, we continued our work and started stability tests and TEV digests.
The TEV digests showed some results so we could detect protein. However, we detected multiple linear TALE proteins instead of circular proteins.
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.
After finishing our experiments, we researched alternative methods that could be used to circularize the TALEs effectively. Some possible changes include:
- 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.
- 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.
