Team Bielefeld sent us a plasmid based on BBa_I746909 having a translation enhancing sequence (5’-UTR). We, the iGEM team Freiburg, sent a plasmid containing coding sequences for turboYFP, a His- and a Halo-Tag. We aimed to analyze if these parts will work in different cell-free protein synthesis environments.

To test the plasmid BBa_I746909 containing a translation enhancing sequence (5'-UTR) we performed a cell-free expression with our own mix and compared it to a sample containing no DNA (negative control).
Both samples were treated alike and compared. All reactions were performed in duplicates. The samples were expressed for 4 hours at 37°C in a 384-well plate using our DiaMIX. After expression, the graph clearly indicates successful expression of GFP. This underlines the ability of the DiaMIX to express vectors from various sources and the effectiveness of a 5'UTR.

Figure 1: Expression of BBa_I746909. The expression was conducted for 4 h and relative fluorescence measured in one minute steps. Mean values and standard deviations were calculated from duplicates.

Stockholm sent us the lysate of a HER2 domain binding affibody with a His-tag as well as a glycerol stock of the E. coli Top10 strain they used for expression and organized the sponsoring of the HER2-antigen from R&D-systems.
Freiburg tried 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 early detection of cancer biomarkers, Stockholm 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 not suitable for an anlysis 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 possibility would have been to spot the His-tagged affibody on a PDITC surface and flush the slide with 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 lysis of the E.coli cells that expressed the affibody. To identify the expressed protein we used Ni-NTA conjugated HRP antibody. Additionally, we stained the membrane with amidoblack to determine successful transfer of the proteins onto the membrane (figure 1). Unfortunately, there was no signal detectable by chemiluminescence at the expected molecular weight. Therefore, we did not proceed with purification.

We contributed to the iGEM Newsletter published by the iGEM Team Amoy.

The iGEM Team Amoy published a regular Newsletters for the iGEM competition together with Paris_Bettencourt and Pasteur_Paris. They provided all iGEM Teams with the possibility to share their project idea, information about experiments they are performing or opinions about critical 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 delighted to be able to contribute to such a great piece of work.

All issues that were published during this year's iGEM competition and further information can be found here.

The iGEM Team Tübingen provided us with a construct coding for the Spy-Tag.

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 SpyCatcher immobilized on top of an activated silane layer. The SpyCatcher would then interact specifically with 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 carboxyfluorescine labeled SpyTag.

Unfortunately, we were not able to establish a SpyCatcher surface and test the corresponding SpyTag we received from Team Tübingen due to time constraints.