Team:Freiburg/Collaborations
Collaboration with iGEM Team Bielefeld
Bielefeld sends a plasmid based on BBa_I746909 that has a translation enhancing sequence (5’-UTR), and Freiburg sends a plasmid containing turboYFP, a His- and a Halo-Tag. We aimed to compare if these parts work in different cell-free protein synthesis environments.
To test the plasmid BBa_I746909 containing a translation enhancing sequence (5'-UTR) we compared it to the GFP constructs we use for cell-free expression (HA-GFP-His6-His6 and His-GFP-Spy). Both plasmids were treated alike and compared to a sample containing no DNA (negative control) and a dilution series of expressed and purified GFP (positive control). All reactions were performed in triplicates. The samples were expressed for 4 hours at 37°C in a 384-well plate using our own lysate and premix. After expression, a western blot and dot blot were performed. The graph clearly indicates the sucessfull expression of GFP from the received vector. This speaks for The ability of our mix to express vectors from various sources and the effective design of the 5 'UTR.
Figure 1: Expression of GFP from Bielfeld received vector. Expression was conducted for 4 h. Measuremnt was taken every minute. Mean values and standarddeiation was calculated from dublicates.
Collaboration with iGEM Team Stockholm
Stockholm sends the lysate of a HER2 domain binding affibody with a His-tag, as well as a glycerol stock of the Top10 bacteria strain they used for expression and organizes the sponsoring of the HER2-antigen from R&D-systems.
Freiburg tries to measure the binding of the purified HER2-antigen to the His-affibody lysate in iRIf.
Figure 1: Amido black stained Western Blot of His-tagged affibody. No signal could be detected at 10 kDA (size of the affibody)
As the iGEM Team Stockholm 2015 also works on a diagnostic tool, we thought it would be great to combine our two approaches. For the early detection of cancer biomarkers they tried to establish an affibody based bacterial biomarker assay (ABBBA). We planed to measure the binding of the purified HER2-antigen to the corresponding His-tagged affibody Stockholm sent us, which we immobilized on our Ni-NTA surface. Unfortunately we realized, that the HER2-antigen was also His-tagged, so it was impossible to measure the binding on a Ni-NTA surface. The HER2-antigen would bind to the surface, precluding a detection of its binding to the affibody.
To circumvent this problem we spotted the HER2-antigen on a PDITC surface and measured the binding of the affibody to the protein. Due to the small size of the affibody (under 10 kDa) it's not possible to observe the binding with iRIf because the detection limit of the system is around 10 kDa.
A third possiblity would have been to spot the His-affibody on a PDITC surface and flush over the antigen. Therefore it would have been necessary to purify the affibody, so we expressed it freshly from the glycerol stock Stockholm sent us. We performed a Western Blot of the pellet and the soluble fraction after the lysis of the E.coli cells that expressed the affibody. To identify the expressed protein we used anti-his conjugated HRP antibody. Additionally we stained the membrane with Amido Black to determine a successful transfer of the proteins onto the membrane (fig.1). Unfortunately there was no signal with the chemilumineszenz detection at the expected molecular weight. Therefore we did not perform the purification.
Collaboration with iGEM Team Amoy
We contributed to the iGEM Newsletter published by the iGEM Team Amoy.
The iGEM Team Amoy published regular Newsletters for the iGEM competition together with Paris_Bettencourt and Pasteur_Paris. It provided all iGEM Teams with the possibility to share their project idea, information about experiments they are performing or opinions about crucial topics of synthetic biology as well as to ask for help with complications they faced during this summer. All in all, there were seven issues published with different contents, three of them being special issues dealing with setting up an iGEM Team, the current situation of synthetic biology and software used in iGEM.
We were asked to contribute to the work of the iGEM Team and used this opportunity to share our project and thoughts with the iGEM community. We were pleased to be able to contribute to such a great piece of work.
All the issues that were published during this year's iGEM competition and further information can be found here.
Collaboration with iGEM Team Tübingen
The iGEM Team Tübingen provided us with a Spy-tagged protein.
For the establishment of a specific surface on the glass slide of the DiaCHIP we followed different approaches. Among others, we planned to test a surface with a SpyCatcher immobilized on top of an activated silane layer. The SpyCatcher would then interact specifically with cell-free expressed Spy-tagged proteins. In order to be able to verify the predicted function of our surface before performing cell-free expression, the iGEM Team Tübingen kindly provided us with a purified carboxyfluorecine labelled SpyTag.
Unfortunately, due to time constraints, we were not able to establish a SpyCatcher surface and test the corresponding SpyTag we received from Team Tübingen.
