Difference between revisions of "Team:Bielefeld-CeBiTec/Design"
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| − | <p> The next step was to create a dark environment. Therefore we did handicrafts to design a | + | <p> The next step was to create a dark environment. Therefore we did handicrafts to design a black carton. Now it was possible to take photos under defined conditions and constant dark background. </p> |
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<figure style="margin:auto; width: 200px"> | <figure style="margin:auto; width: 200px"> | ||
| − | <a href="https://static.igem.org/mediawiki/2015/1/11/Bielefeld-CeBiTec_black_box.png" data-lightbox="detection" data-title=" The black | + | <a href="https://static.igem.org/mediawiki/2015/1/11/Bielefeld-CeBiTec_black_box.png" data-lightbox="detection" data-title=" The black carton: tinkered to take photos under same conditions"><img src="https://static.igem.org/mediawiki/2015/1/11/Bielefeld-CeBiTec_black_box.png" ></a> |
<figcaption> The black box: tinkered to take photos under same conditions | <figcaption> The black box: tinkered to take photos under same conditions | ||
</figcaption> | </figcaption> | ||
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<figure><center> | <figure><center> | ||
<img src="https://static.igem.org/mediawiki/2015/e/e0/Bielefeld-CeBiTec_app3small.png"> | <img src="https://static.igem.org/mediawiki/2015/e/e0/Bielefeld-CeBiTec_app3small.png"> | ||
| − | <figcaption>The application is adapted to the | + | <figcaption>The application is adapted to the case shown in the picture. The black case provides the ideal environment for capturing pictures under reproducible conditions. The application will calculate whether a contamination is present and give out an list of the contaminants in the water sample.</figcaption></center> |
</figure> | </figure> | ||
</div> | </div> | ||
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<div class="The 3D print"> | <div class="The 3D print"> | ||
<h2> The 3D print </h2> | <h2> The 3D print </h2> | ||
| − | <p> Ok, the fluorescence detections works fine, but it's not really practical to take the photo. The filter have to be in the right position direct in front of the camera respectively of the flash. Also it has to be quite dark for high quality pictures. Therefore we designed a | + | <p> Ok, the fluorescence detections works fine, but it's not really practical to take the photo. The filter have to be in the right position direct in front of the camera respectively of the flash. Also it has to be quite dark for high quality pictures. Therefore we designed a black case and realized it with a 3D printer. Marco Radukic designed the case in exact accordance with our guidline. The top can be changed specific for the smartphone you use. The test stripe can be placed on the push loading drawer and inserted into the box. So it's quite easy to use. You just have to put the smartphone on the top, insert the test strip an take the photo. </p> |
<figure style="margin:auto; width: 600px"> | <figure style="margin:auto; width: 600px"> | ||
| − | <a href="https://static.igem.org/mediawiki/2015/9/98/Bielefeld-CeBiTec_3D_box_modell.png" data-lightbox="detection" data-title=" | + | <a href="https://static.igem.org/mediawiki/2015/9/98/Bielefeld-CeBiTec_3D_box_modell.png" data-lightbox="detection" data-title="On the left side you can see the 3D modell of the case, realized by Macro Radukic in exact accordance with our guidline. On the right you can see the printed black case. Printed from Thomas Schäfer with his 3D printer "><img src="https://static.igem.org/mediawiki/2015/9/98/Bielefeld-CeBiTec_3D_box_modell.png" ></a> |
| − | <figcaption> | + | <figcaption> On the left side you can see the 3D modell of the case, realized by Macro Radukic in exact accordance with our guidline. On the right you can see the printed black case. Printed from Thomas Schäfer with his 3D printer |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
Revision as of 17:49, 18 September 2015
Output Signal processing
A Prototyp for easy fluorescence detection
The problem
We planed to design a practical test strip using fluorescence as output signal. Green fluorescent protein (GFP) is among the most frequently used reporter proteins and has been used in numerous iGEM projects. But detecting fluorescence outside the lab sounded impractical to us. So we had to find an easy way to analyze the output.
The first steps
Before testing we decided to measure the extinction and emission spectra from sfGFP as a basis to choose the most promising filter combination and to have a look if the smartphone flash really excites sfGFP.
In the beginning of our project, we wanted to test if it could work to photograph fluorescence with a smartphone. We got a filter from light engineering from the women cultural center Bielefeld e.V. ("Frauenkulturzentrum Bielefeld e.V.") and put it in front of the smartphone. In the picture below, we took a picture with these filter from purified GFP, sfGFP lysate and lysate without sfGFP. You can see different colors and a different brightnesses in the picture. So in our first impression we thought it's worth to try some other filters, to find the perfect filter for sfGFP imaging with almost no background signal.
Therefore we bought a lee color filter catalog and began to test different filter combinations. The preselection was possible, because we had access to the light transmitting spectra of almost every filter.
The next step was to create a dark environment. Therefore we did handicrafts to design a black carton. Now it was possible to take photos under defined conditions and constant dark background.
The filter combinations
The essential requirements for taking comparable photos are done. Now we had to find the ideal filter combination. So we made a lot of photos with the preselection. We tested 6 filters for emission and combined every filter for emission with up to 16 filters for extinction.After analyzing the photos with the image processing software Fiji, the optimal filter combination found was tokyo blue (071) in front of the flash and twickenham green (736) in front of the camera.
As you can see in the picture above it is really important to choose the right filters to get a high fluorescence signal and a low background signal. But is it only possible to photograph sfGFP and GFP? To find it out, we tried to photograph monomeric red fluorescent protein (mRFP) lysate, as well.
Does fluorescence photography work on Paper?
So far so good. Fluorescence imaging of liquids in quite high amounts is possible. But does it also works with small volumes and on paper? Therefore we took normal laboratory filter papers. (MN 827 B from Macherey and Nagel, C 350 L and FN3 from Munktell and a laboratory filter paper we got from Merck) and put the lysates on the paper and took the photos. Exemplary the results from laboratory filter paper from Merck are shown. The other ones showed the same results. As you can see in the picture below this paragraph, it is possible to photograph volumes of 5 µl purified GFP up to a concentration of 17,5 mmol/L.
Now we wanted to find out if it is possible to photograph the fluorescence from a CFPS experiment on paper. So after a CFPS run on paper, we took the scrap of paper and took a photo from it. As you can see in the picture below.
The App
Now it's possible to photograph fluorescence, but sometimes it's difficult to see the differences. And analyzing the pictures with the image processing software Fiji requires know-how of the user. On the one hand for the program and on the other hand for interpreting the results. We solved this problem with a smartphone App. We coded the App with Android Studio 1.2.2 and can be installed on smartphones on Android 4.2 (Jelly bean) and later. The App determines the median of the greenvalue of a area of pixels in the sensorspot and compares it to the pixels of the negative control (the not induced sensor). If the ratio of a sample and the negative control is above a certain theshold (10%), valid biosensor signal is detected. Additionally it is checked whether the value for the positive control (drop of sfGFP) is above a certain value, thereby confirming the functionality of the biosensor. Furthermore the App displays specific information regarding the different heavymetals and date rape drugs to inform the user. You can download it by clicking on the button below. You need to unzip the file on your phone, since one cannot upload .apk files. Alternatively download it here and delete the fileextension ".txt".
The 3D print
Ok, the fluorescence detections works fine, but it's not really practical to take the photo. The filter have to be in the right position direct in front of the camera respectively of the flash. Also it has to be quite dark for high quality pictures. Therefore we designed a black case and realized it with a 3D printer. Marco Radukic designed the case in exact accordance with our guidline. The top can be changed specific for the smartphone you use. The test stripe can be placed on the push loading drawer and inserted into the box. So it's quite easy to use. You just have to put the smartphone on the top, insert the test strip an take the photo.
