Team:Freiburg/Results
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?
Ich finde irgendwie den Namen "Main results" irgendwie... zu schwach. Einfach mal ein paar in den Raumge worfen - ob ihrs ändert is geschmackssache: Highlights, key results, essentials, essential results, Milestones (ps1309)
Essential Results
Our aim was to develop a novel diagnostic tool enabling fast, simultaneous and label-free detection of antibodies in human blood sera. We successfully generated ou own cell-free expression system based on an E. coli lysate which revealed an expression efficiency comparable to a commercial kit. To immobilize the expressed proteins, we established a surface specifically binding our target proteins. This system was used to produce a protein array that can be flushed with a serum sample. The binding of antibodies is detected in a label-free manner by an interferrometric method called iRIf. Based on the interference of light reflected at different thin layers, this method allows real-time detection. Moreover, we succeeded to rebuilt the detection device in a simplified and cost efficient variant and thus made it available for future iGEM Teams. Using the DiaCHIP, we did not only show the presence of antibodies in distinct solutions but also verified them in complex samples as human blood serum.
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. By comparing the spotted pattern with the localization of signal as detected by iRIf, the systems specificity even for complex samples was shown. ▼ Detailed Description.
The antigen used for detection of anti-tetanus antibodies is derived from the tetanus neurotoxin and commonly used for in vitro testing. For preparation of the DiaCHIP, this antigen had to be overexpressed in Escherichia coli and purified from the whole cell lysate. To verify successful purification of the antigen Western Blot analysis was performed (fig. 1). Using an antibody targeting the N-terminal His-tag, revealed a protein with a molecular weight of about 50 kDa. This correlates to the expected molecular weight of the antigen (Reference). The purified antigen was immobilized on a Ni-NTA surface that specifically binds His-tagged proteins. Look here to read more about the establishment of this surface. As a positive control, self-purified, His-tagged GFP was immobilized at another spot. The reliable detection of anti-GFP was shown before (link). No binding event should be detected at the negative control spot that was covered with bBSA/mCherry (??).
The human serum samples we obtained have been taken before and three? weeks after a patient received a vaccination boost against tetanus. Analysis of the samples was performed using the same array set-up. Figures 2 and 3 show quotion pictures representing changes in the thickness of the slide while it was flushed with the respective samples. The negative control spots do not indicate any binding events, while the binding of anti-GFP to the positive control spot verifies that both experiments are reliable. Comparison of the antigen spots shows that we specifically detected anti-tetanus antibodies in human serum in response to vaccination. No binding event was detected by flushing the array with the sample assumed to be negative (fig. 2), whereas a distinct spot is visible after flushing with the positive sample (fig. 3). Both measurements in real-time are shown in parallel in video 1.
The increase in relative light intensity at distinct spots over the course of the experiment is visualized in figure 4 and can be correlated to the amount of antibody binding to the respective spot. This indicates that the DiaCHIP enables quantification of antibody titers in addition to detecting their presence.
Besides tetanus, other antigens of immunological relevance were taken into account. See all the results we obtained in terms of diagnostics.
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
Our self-made cell-free expression mix, in combination with specific glass surfaces, allows for the expression and purification of target proteins in a simple manner. The mix, after expressing His-tagged GFP, was spotted on a Ni-NTA coated glass slide. After several washing steps it could be shown, that the proteins maintain their antibody binding properties even in complex samples. Therefore serum of a rabbit, immunized against GFP, was flushed over the slide and a bspecific binding could be detected in iRIf. Even the spot showing the lowest signal for cell-free expressed GFP was significantly brighter than the negative control provided on the same slide. ▼ 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 NTA solution over night and loading with Nickel-ions. The slide was blocked with a solution of 5 mg/mL BSA and 5 % ethanolamine in PBS and cell-free expressed GFP with a His tag, a positive control as well as a negative control (Dia MIX without DNA) 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 as 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.
Commercial systems using the iRIf-technology (imaging reflectomretric interference) are mostly bulky and expensive machines even though the physics which they are based on are rather simple. So we decided on building our own device sonsisting of not much more than two lenses and a camera. With this system we were able to reliably detect the binding of anti-GFP to GFP, thus confirming a detection sensitivity in the range of protein-protein interactions. To enable future iGEM teams to profit from this device as we did, we provide all plans and parts necessary to rebuild it, on this website for everyonbe to download and use. ▼ Detailed Description.
To test our device we aimed at reproducing measurements that we were previously able to perform in our commercial device. For this reason we focused on using the two antibodies: anti-GFP and polyclonal anti-rabbit. GFP and rabbit derived anti-HCV (hepatitis C virus) antibodies were used as antigens in this experiment. Note that the anti-HCV antibodies only served as binding partners for the anti-rabbit antibodies, since no HCV proteins were used in this experiment. The antigens were spotted onto an iRIf slide whose binding layer consisted of an APTES/PDITC surface. The spots on the slide were made by pipetting 3,5 µl (1 mg/ml) rabbit-anti-HCV protein and 3,5 µl (1 mg/ml) GFP onto the slide and incubated over Night. After incubation, the slide was blocked for 30 min in BSA solution. A Canon 50D camera was used to record the measurement and was set to take one picture every 5 seconds. The exposure time was set so that the pixels in the image were approx. 80% of maximum light saturation before the solution was flown onto the chip. The antibody solutions were pipetted into the flow-chamber without the use of any microfluidics device. Instead a syringe was loaded with 660 µl [5 µg/ml] anti-GFP solution (diluted in PBS) and connected to the input tube of our device. The content of the syringe was then slowly released into the binding chamber of the device by gently dispensing the solution from the syringe. When the whole volume ran over the chip, the process was repeated with 660 µl [5 µg/ml] anti-rabbit antibody in the same way. The injection of both solutions took approximately 45 minutes.
Video 1 shows the results of the measurement. Both binding of anti-GFP and anti-rabbit to the corresponding antigen spots could be observed. Due to the fact that the experiment was performed on our demonstration device which is build with a transparent casing, fluctuations in surrounding light had a strong, detrimental influence on the measurement quality. To minimize the influence of the surrounding light being unstable, the resulting pictures had to be averaged over 10 pictures each. This in turn lead to a more stable light situation, however the signal strengh dropped as a consequence. Figure XY shows a quotient picture during the measurement where the light situation was temporarily appropriate. The problem of surrounding light scattering into the device can of course be overcome using a non-transparent casing.
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.
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.
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.
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.
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
