Team:Freiburg/Results
Vll etwas doofer Kommentar, aber die "Detailed Description" buttons sehen aus wie links. Ich hab da erst gar nicht drauf geklickt, da ich dachte da werde ich wo ganz anders hingeleitet. Wenn jemand ne kluge Idee hat, wie man andeuten kann, dass da ein drop-down menü drunter liegt, wre das cool (ps1209) Besser?! (jb1209)
Nicole: Hab ich es richtig verstanden, dass hier nur eure Highlights and Ergebnissen dargestellt werden sollen (mit verlinkung zu den anderen Ergebnissen)? Was haltet ihr von einer kurzen Einleitung... In the last months we (aimed to) developed a diagnostic tool which enables [...] We succeded to detect anti-tetanus antibodies in human serum, develop/generate our own cell-free mix...
mehr Formulierungen wie: we demonstrated..., we (successfully) tested, we achieved... Stefan: Ihr sprecht hier immer von spotted, dabei wird aber nicht klar wie ihr das gemacht habt. Vielleicht sollte man immer dazu schreiben spotted by hand. Was denkt ihr dazu?
Main Results
Detection of anti-Tetanus antibodies in human blood serum
We specifically detected anti-Tetanus antibodies in human blood serum. To test the DiaCHIP under real-life conditions, we analyzed the blood serum of a vaccinated person for the presence of anti-Tetanus antibodies. We compared blood samples before and after vaccination and could directly detect its effect. To capture the antibodies, the corresponding antigen was expressed in E. coli, purified by His-tag affinity purification and spotted on a specific Ni-NTA surface. ▼ Detailed Description.
Applying the sample taken three weeks after vaccination to the DiaCHIP resulted in a significant signal at the antigen spot, whereas no signal was obtained with the sample assumed to be negative. But convince yourself of how great the DiaCHIP performed. The video on the right shows these measurements right next to each other in real-time. Hier noch mehr Results beschreibung? Wie viel Blut, welche Verdünnung, welche Flussraten? Bindekurve? Ganztifizieren des Signals könnte vor allem für dieses Exp sehr interessant sein. (Stefan)
Besides tetanus, some other antigens of immunological relevance were taken into account. See all the results we obtained in terms of diagnostics, including an approach for quantification of the measurements.
Auch, dass euer Mix low-cost ist, und trotzdem auf dem Niveau (oder besser) eines kommerziellen kits liegt - Community gedanke etc. (ps12092015)
Detection of anti-GFP antibodies in rabbit serum using cell-free expressed GFP
To show that cell-free expressed proteins can be immobilized on the protein surface while maintaining their antibody binding properties, the expression mix was spotted on a specific Ni-NTA surface after GFP expression. Cell-free expressed GFP was spotted on a specific Ni-NTA slide and flushed with serum of a rabbit immunized against GFP. The lowest spot consisting of cell-free expressed GFP shows a significant signal compared to the negative control (middle spot). ▼ Detailed Description.
We got hold of a more or less 20 years old serum of a rabbit that was immunized with GFP. This gave us the perfect opportunity to test our device with a more complex solution than antibody-dilutions.
For this experiment we used a specific Ni-NTA surface and spotted cell-free expressed GFP on the surface.
For verification of correct expression of GFP with our Dia-Mix we performed a Western Blot analysis (fig. 1). With this we could prove that the cell-free expression was successful.
To obtain the specific surface an iRif slide was plasma activated and incubated with APTES and PDITC. Afterwards the Ni-NTA layer was added via incubation of the slide in Ni-NTA solution over night. The slide was blocked with blocking solution (?) and cell-free expressed GFP with a His tag ( + other GFPs?) and a positive control as well as a negative control (His-mCherry) were spotted onto the surface.
The serum of the GFP-immunized rabbit was then analysed in an iRif measurement. Binding of the GFP-antibodies present in the rabbit serum can clearly be seen, as the respective spots show a strong signal (fig. 1). A parallel measurement with serum of a rabbit that was not immunized (fig.2) does not show any binding events at the GFP spot.
With this experiment we could show that it is possible to analyse complex fluids like serum. In addition to that we demonstrated that our cell-free expressed GFP with a His-tag worked at binding partner of GFP-antibodies. Thus, His-tagged GFP was successfully expressed and immobilized on the surface, still enabling anti-GFP antibodies in a serum sample to bind.
Find out more about the preparation of the DiaCHIP by producing a protein microarray from a DNA template using cell-free expression.
Building our very own, low-cost DiaCHIP measuring device.
The measuring device we used in collaboration with the AG Roth (ZBSA) to detect antibody binding events is a really expensive machine, but the physics which it is based on is rather simple. Our self-built device consistent of not much more than two lenses and a camera (A) reliably detected the binding of anti-GFP to GFP (B). To enable future iGEM teams to profit from this label-free detection method as we did, a cost-efficient and easy to rebuild variant of the device was developed (figure 2A). ▼ Detailed Description.
Figure 2B shows the binding of anti-GFP to GFP as it was established before, measured with our own device. Hier müsste man dann auch noch mehr zum experiment schreiben. Microfluidic von hand. Bildkorrektur erwähnen, wie gemacht? Look here, to see how you can build your own device for label-free detection of antibody binding on a protein microarray needing two lenses, a camera and a little bit of fine feeling (??) sensitivety/sensitiveness/finesse/sure instincts.
Specific surfaces
Since there is a variety of proteins present in the cell-free mix a specific surface on the future protein array is essential. That's why we developed our own Ni-NTA surface for immobilization of the antigens on the chip. All expression constructs contain a His-tag fused to the coding sequence, resulting in antigens that can bind specifically to our Ni-NTA surface. Compared to an unspecific surface (PDITC) we could show that this Ni-NTA surface allows efficient binding of target protein and prevents unspecific binding of other proteins that are part of the cell-free mix. Detailed description.
For cell-free protein expression lots of different proteins, like RNA-Polymerase, ribosomes and other E. coli proteins, are essential. So during the process of cell-free expression all these proteins are, next to the target protein, also present in the microfluidic chamber. To get a sufficient amount of target protein, we therefore established a specific surface chemistry on the glass slide. Next to other tag systems ( link ) we established a Ni-NTA surface because it worked best for us. Using a Ni-NTA covered glass slide (figure ___) we could increase the amount of bound cell-free expressed GFP compared to an unspecific surface (figure___). In both experiments ____2,5 µg?!?____ purified His-GFP were used as positive control. A cell-free reaction performed without DNA, that is supposed to result in no target protein, served as negative control. The cell free reactions were performed for ___2?!___ hours at 37°C. The samples were spotted on the surfaces and incubated on slide o/n. In iRIf, the optical detection method we used, the slides were blocked with BSA and then flushed with anti-GFP antibodies. An increase in light intensity on the slide, where the spotted protein is located, represents a binding event with anti-GFP antibodies. Due to less unspecific binding, interaction of anti-GFP with cell free expressed His-GFP is higher on the Ni-NTA surface. hier Bilder einfügen: https://2015.igem.org/File:Freiburg_GFP_CF_NiNTA.png , Bildunterschrift: Cell-free expressed His-GFP spotted on a Ni-NTA slide. Cell-free expression was performed for 2?!? hours at 37°C. The positive control was purified His-GFP, the negative control was a sample of cell-free reaction performed without DNA. https://2015.igem.org/File:Freiburg_GFP_CF_NiNTA.png Bildunterschrift: Cell-free expressed His-GFP spotted on a PDITC-NTA slide. Cell-free expression was performed for 2?!? hours at 37°C. The positive control was purified His-GFP, the negative control was a sample of cell-free reaction performed without DNA.
Own cell-free mix
The mechanism we are using to copy a DNA array template into a protein array on demand is based on cell-free expression. During our project we successfully established our own cell-free expression system, the DiaMIX, from scratch, starting with an E. coli lysate. With this mix we succeeded in expressing correctly folded GFP and luciferase. Comparison of the DiaMIX with a commercial cell-free expression kit revealed the great potential of our system! ▼ Detailed Description.
Our DiaMIX was prepared based on a protocol of ??? (Ref.). It is a very complex system containing many different enzymes and chemicals for special purposes. An overview of cell-free expression systems and its components can be found on methodology page . To investigate the efficiency of our self-prepared expression system we performed an experiment comparing it with a commercially available kit. In order to obtain reliable results we used the exact same vector, containing a sequence encoding GFP. Like this we were able to trace the amount of expressed GFP using a plate reader over a period of two hours. As a negative control, both mixes were treated equally in a second sample but did not contain DNA. The evaluation is shown in Figure 1 and is based on triplicates. The data was normalized to the mean value of the measurement of air. The uncertainty was calculated using the standard deviation. picture; Figure 1: Cell-free expression of GFP using the DiaMIX and a commercial kit. The reaction was performed in a volume of 50µl each and monitored every minute at 37°C over a period of two hours. Excitation at 480nm, emission at 520nm. Both expression systems were shown to successfully express the applied vector. There is a clear enhancement of light emission observable at 520nm for both mixes in comparison with the respective negative control. The expression of GFP using the DiaMIX seems to be slightly better than by using the commercial kit. Still, one has to mention the actual purpose of the commercial kit to express linear templates. However, as the basis protocol for our mix was optimized for circular templates, we used a circular vector for the experiment. Furthermore, as already shown above, we successfully verified the expression of GFP with our own cell-free mix in an iRIf measurement (LINK?!). To obtain these final results, a lot of optimization and testing had to be done. If you want to know more about how we established the DiaMIX for the DiaCHIP, you can find detailed information about the most important experiments on our results page .
Explore the DiaCHIP
For establishing our device, we optimized all steps from the immobilization of DNA to the top of the flow-chamber until the specific binding of the target proteins on the glass slide. This way, we generated protein arrays we could use to detect different antibodies in human and rabbit serum with the iRIf-detection method.
Click on the images below to explore our experiments from expression to detection!
Assembling the DiaCHIP
Diagnosis of Antigens
Click on one of the images to get further insight how we build up our DiaCHIP
