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+ | <h2> Construction </h2> | ||
+ | <h3> Purification column EluX </h3> | ||
+ | <article> | ||
+ | For the construction of our purification column, we aimed for two different approaches. The first one was to build the column on our own, using a 3D-Printer to get the basic parts of the column. This was important, as we wished to provide the construction files to the iGEM community and enable other teams to use and improve our work. The second approach was to construct this column in a more professional way, and so we asked the technical workshop of our university for assistance. They helped us by cutting the parts more precisely and so improve the applicability of the column. This lead us to two versions of the purification column, a 3D-printed show model and the application model.</article> | ||
+ | <h4> 3D-Printed show model </h4> | ||
+ | <article> | ||
+ | We used a 3D-Printer to print the scaffold blocks and cut the front and back plate and the top and bottom hold bars by our self from acrylic glass. The glass slides were not biotinylated yet and the plates are not UV-permeable, so not usable, but the basis of further improvement and test of sealing and compatibility of the lab pumps. | ||
+ | </article> | ||
+ | <div class="figure small"> | ||
+ | <img class="figure image" src="https://static.igem.org/mediawiki/2017/1/12/T--Bielefeld-CeBiTec--YKE_3D_printed_parts_for_the_column.png"> | ||
+ | <p class="figure subtitle"><b>Figure 8: 3D-printed parts of the purification column.</b> | ||
+ | </div> | ||
+ | <article> | ||
+ | Besides the 3D-printed parts we cut and drilled the acrylic glass blocks and plates, the 3-mm threaded rods and the rubber mats at the home of a team member following our 3D-model. | ||
+ | </article><div class="figure small"> | ||
+ | <img class="figure image" src="https://static.igem.org/mediawiki/2017/7/70/T--Bielefeld-CeBiTec--YKE_markus_bohrend_for_the_column.jpg"> | ||
+ | <p class="figure subtitle"><b>Figure 9: Preparation of the non-3D-printed parts at the home of Team member Markus.</b> | ||
+ | </div> | ||
+ | <article> | ||
+ | We then assembled the parts and luckily everything matched as expected. The hand cut pattern of the rubber mat was still a bit uneven but should be still functional for some preliminary tests. | ||
+ | </article> | ||
+ | <div class="figure medium"> | ||
+ | <img class="figure image" src="https://static.igem.org/mediawiki/2017/c/c0/T--Bielefeld-CeBiTec--showmodels_inside_outside.jpg"> | ||
+ | <p class="figure subtitle"><b>Figure 10: Inside and outside of the EluX prototype.</b> | ||
+ | </div> | ||
+ | <article> | ||
+ | We were very happy that our first self-build model looks exactly like the 3D-modell and we were very excited to show it to some experts to gain some opinions and advices for the following design and construction of the application model. | ||
+ | </article> | ||
+ | <div class="figure medium"> | ||
+ | <img class="figure image" src="https://static.igem.org/mediawiki/2017/f/f8/T--Bielefeld-CeBiTec--3D_compare.jpg"> | ||
+ | <p class="figure subtitle"><b>Figure 11: Comparison of the build and presented show model and the 3D-Model.</b> | ||
+ | </div> | ||
+ | <article> | ||
+ | We used this show model at our meetings with Prof. Dr. Dirk Lütkemeyer, General Manager of BIBITEC GmbH, and Dr. Benjamin Müller, CEO of Biofidus AG for Analytical Services. They advised us in building the shown models professionally to test and validate the system and also discussed the further development of the column and possible applications. | ||
+ | </article> | ||
+ | </div> | ||
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Revision as of 23:48, 26 August 2017
Overview
Purification Column EluX
To verify the elution as easily as possible, we created a fusion protein containing GFP for a visible fluorescence signal and streptavidin to immobilize the protein in our column using biotin. The two protein parts are connected through a flexible glycine-glycine-serine-linker of eleven amino acids, in which the sixth amino acid is our 2-NPA. This ncAA will then induce the cleavage of our target protein (GFP) from the streptavidin compound in the purification process.
Development
Purification column EluX
![](https://static.igem.org/mediawiki/2017/d/d4/T--Bielefeld-CeBiTec--YKE_Development_of_the_column.png)
Figure 1: The six steps in the development process of the purification column
A) BSA-coated micro well plate, B) Hollow cylindrical column, C) Biotinylated microscope slides, D) First flow model, E) second flow model, F) future concept of a microfluidic like column.
BSA-coated micro well plate
![](https://static.igem.org/mediawiki/2017/f/f3/T--Bielefeld-CeBiTec--YKE_microwell_plate_for_the_column.png)
Figure 2: Concept of a microwell plate coated with biotinylated BSA.
Cylindrical column
![](https://static.igem.org/mediawiki/2017/f/f8/T--Bielefeld-CeBiTec--YKE_cylindrical_model_of_the_column.png)
Figure 3: Concept of our cylindrical column
A)3D-Modell of a hollow column with an LED-rod in the middle and a carrier material coated with biotinylated BSA as first concept of a purification system using 2-NPA.
Biotinylated glass slide
![](https://static.igem.org/mediawiki/2017/7/79/T--Bielefeld-CeBiTec--YKE_microscope_slides_for_the_column.png)
Figure 4: Three microscope glass slides as basis for biotinylated surfaces for the purification column.
First flow model
![](https://static.igem.org/mediawiki/2017/5/5f/T--Bielefeld-CeBiTec--YKE_EluX_prototype_1_for_the_column.png)
Figure 5: EluX prototype one.
First flow model of a purification column with parallel biotinylated glass slides inside and UV-permeable acrylic glass plates in front and back to enable the radiation with light of 365 nm wave length.
Second flow model
![](https://static.igem.org/mediawiki/2017/2/2f/T--Bielefeld-CeBiTec--YKE_EluX_prototype_2_for_the_column.png)
Figure 6: EluX prototype two.
Second flow model of a purification column as basis for a workshop and a 3D-printed model to test, evaluate and improve the concept of light induced elution.
Future microfluidic / flow-bundle model
![](https://static.igem.org/mediawiki/2017/7/7c/T--Bielefeld-CeBiTec--YKE_future_predicitions_for_the_column.png)
Figure 7: Future concept of EluX.
Concept of a microfluidic like (a) and a bundle (b) purification column designed with the support of Dr. Benjamin Müller and Prof. Dr. Dirk Lütkemeyer as future prediction of how the procedure of light induced elution could be used.
Construction
Purification column EluX
3D-Printed show model
![](https://static.igem.org/mediawiki/2017/1/12/T--Bielefeld-CeBiTec--YKE_3D_printed_parts_for_the_column.png)
Figure 8: 3D-printed parts of the purification column.
![](https://static.igem.org/mediawiki/2017/7/70/T--Bielefeld-CeBiTec--YKE_markus_bohrend_for_the_column.jpg)
Figure 9: Preparation of the non-3D-printed parts at the home of Team member Markus.
![](https://static.igem.org/mediawiki/2017/c/c0/T--Bielefeld-CeBiTec--showmodels_inside_outside.jpg)
Figure 10: Inside and outside of the EluX prototype.
![](https://static.igem.org/mediawiki/2017/f/f8/T--Bielefeld-CeBiTec--3D_compare.jpg)
Figure 11: Comparison of the build and presented show model and the 3D-Model.