Difference between revisions of "Team:Grenoble-Alpes/Demonstrate"
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| − | <p>If any detail is needed about the fluorescence sensor, you can find some explanations < | + | <p>If any detail is needed about the fluorescence sensor, you can find some explanations <a href="https://2018.igem.org/Team:Grenoble-Alpes/fluorescence_module" style="font-weight: bold;">here</a>. </p> |
Revision as of 21:19, 15 October 2018
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DEMONSTRATE
The following page contains the results and succes that we actually obtained with our system.
Tests were performed on a biological transformation because we knew that the tricky part would be the cooling step of the thermal shock. In fact, the module could only go down to around 11 degrees so we looked out for literature about optimization of the transformation.
If any detail is needed about the fluorescence sensor, you can find some explanations here.
To prove that the fluorescence unit is efficient enough to detect fluorescence in the device, we followed kinetically a transformation process to see the fluorescence evolution. We were able to capture fluorescence after 13h of incubation. The following video displays the fluorescence evolution in the sample over 19h of experimentation.
VIDEOThe time given at the top left of the picture corresponds to the delay after the beginning of the experiment. The sample seems to move as it is shaken in the incubator and the block where is this sample is not connected to the camera block.
To perform this experiment, we transformed 50 µL of competent Top10 with 2 µL of DNA. The thermal shock was realized in our aluminum blocks - one was set to 42°C and the other one to 13°C -, this to show that the thermal shock that we established in our device leads to a successful fluorescence analysis. However, the incubations are realized in a real incubator, as we lacked time to make sure that we could incubate in the device. Moreover, a cover to limit the evaporation of the sample would certainly have been necessary. Finally, the proof of concept was performed with the test prototype. It works exactly as the unit on the device, but it cannot be used on the device.
To follow kinetically the process, pictures were taken each hour with the Picamera and saved in the Raspberry Pi. As you can see in the GIF, fluorescence is obvious after 14h of experimentation and its intensity starts to decrease after 16h of experimentation. Several factors can explain this phenomenon:
Fluorescence quenching by the time. Fluorescence decreases very fast, so it cannot be the main reason, but it certainly takes part in the decrease of fluorescence intensity
The evaporation. The Eppendorf must be open to perform the fluorescence measurement, and Parafilm significantly reduces the contrast of the pictures. The sample did not evaporate enough to reduce the pictured surface, but the top of the sample is a little further from the camera.
Fluorescence quenching due to bacterial death. The bacteria keeps growing in the culture medium but by the time the tube lacks nutrients, leading to the death of the bacteria. It must be the main reason as a precipitate could be observed in the Eppendorf at the end of the experiment.
Another point that should catch your attention is the fluorescence intensity which is much brighter than on the pictures in the fluorescence unit page. We thought the illumination conditions were the same as for the calibration curve, which is obviously wrong. It does not call into question the proof of concept, but it would have been interesting to evaluate the bacterial concentration after a certain time.
In a nutshell, we proved that the unit is working well but we were not able to exploit the experiment at its most. The fluorescence unit is able to detect fluorescence after a transformation process realized in our device, 13h at least are necessary to perform an analysis. Unfortunately, we could not evaluate the bacterial concentration using the calibration curve as the illumination condition were not the same.